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R W van Leen

Publications and source records attributed to R W van Leen.

11 recordsLinked to original sources
PubMed1991

Receptor and antibody interactions of human interleukin-3 characterized by mutational analysis.

Human interleukin-3 (hIL-3) is a regulator of proliferation and differentiation of multipotent hemopoietic progenitor cells. Mutants of hIL-3 have been constructed by oligonucleotide-directed mutagenesis and expressed in Escherichia coli and Bacillus licheniformis. Purified muteins were assayed for induction of DNA synthesis in IL-3-dependent human cells and for binding to the IL-3 receptor. Residues at the NH2 and COOH termini together comprising one-quarter of the molecule could be removed without loss of biological function. Deletions of 6-15 residues within the central part of the molecule caused a large reduction (up to 5 logs) but no complete loss of activity. Substitution of evolutionary conserved residues resulted in a strong decrease of biological activity and demonstrated that the S-S bridge is an essential structural element in hIL-3. Interestingly, four muteins displayed a significantly higher potency of binding to the IL-3 receptor than in stimulating DNA synthesis. These results demonstrate that receptor binding may be (partly) disconnected from activation of DNA synthesis. Analysis of hIL-3 muteins demonstrated that the majority of monoclonal antibodies are directed against a small portion of the IL-3 molecule. The neutralizing potential of individual monoclonal antibodies could be increased by a combination of antibodies directed against nonoverlapping epitopes.

Amino Acid Sequence↗
PubMed1991

Production of human interleukin-3 using industrial microorganisms.

We expressed a cDNA encoding the multicolony stimulating factor interleukin-3 in a variety of cell types, including bacteria, yeast and mammalian cells. After evaluation of the advantages and disadvantages of each potential system, we designed a production and purification scheme using Bacillus licheniformis. The purification consists of hydrophobic interaction chromatography, two steps of ion exchange chromatography and gel filtration. The purified and formulated product entered clinical trials in November 1989.

Animals↗
PubMed1990

Species specificity of human interleukin-3 demonstrated by cloning and expression of the homologous rhesus monkey (Macaca mulatta) gene.

To enable preclinical studies on homologous interleukin-3 (IL-3) in primate species, we isolated the gene encoding Rhesus monkey IL-3 (RhIL-3). The nucleotide sequence of the RhIL-3 gene displayed 92.9% homology with that of the human IL-3 (hIL-3) gene. The isolated RhIL-3 gene encodes a 143-amino acid (aa) precursor polypeptide, nine C-terminal residues shorter than the human protein. Protein homology was found to be 89.5% for the signal peptide (19 aa) and 80.5% for the mature protein (124 aa). Comparison of the human and RhIL-3 coding sequences showed that the majority of substitutions had occurred at amino acid replacement sites indicating a rapid evolution of the IL-3 protein. After expression of a genomic fragment in COS cells, RhIL-3 cDNA was constructed, which enabled large-scale production of the RhIL-3 polypeptide, RhIL-3 produced by Bacillus licheniformis and purified to homogeneity appeared to be approximately 100-fold more effective in stimulating Rhesus monkey hematopoietic progenitors than hIL-3, whereas RhIL-3 and hIL-3 showed comparable stimulatory activity on normal as well as malignant human hematopoietic cells. Thus, the rapid evolution of hIL-3 has resulted in a unidirectional species specificity, which most likely restricts the in vivo effects of hIL-3 in Macaca species.

Amino Acid Sequence↗
PubMed1990

Highly increased production of bone marrow-derived blood cells by administration of homologous interleukin-3 to rhesus monkeys.

Recombinant rhesus monkey interleukin-3 (IL-3) was administered to normal rhesus monkeys in graded doses ranging from 3 to 30 micrograms/kg/d subcutaneously for 30 consecutive days or given as a continuous intravenous infusion at a dose of 30 micrograms/kg/d for 16 days. After a lag phase of about 1 week, a highly increased, dose-dependent production of bone marrow-derived blood cells was observed, preceded by amplification of bone marrow hematopoietic progenitor cells. Simultaneously, peripheral blood progenitor cells rose. The increase included basophilic, eosinophilic and neutrophilic granulocytes, monocytes, and the erythrocyte and platelet lineages. Characteristically, a T-lymphocyte response was absent. It is concluded that IL-3 in vivo stimulates blood cell production from an immature, multipotent progenitor cell.

Animals↗
PubMed1990

Interleukin-3.

Interleukin-3 (IL-3) is a hemopoietic growth factor involved in the survival, proliferation and differentiation of multipotent hemopoietic cells. In five mammalian species, including man, the gene encoding IL-3 has been isolated and expressed to yield the mature recombinant proteins. The human IL-3 gene encodes a protein of 133 amino acids with two conserved cysteine residues and 2 potential N-linked glycosylation sites; human native IL-3 has not been characterized. Comparison of the IL-3 genes revealed a more rapid evolutionary divergence than has been observed for other hemopoietic growth factors, and, hence, a more pronounced species specificity of the functional proteins was found. In agreement with its stimulatory action on immature multipotent cells, the in vivo actions of homologous recombinant IL-3 in nonhuman primates include a highly increased production of blood cells along the neutrophilic, eosinophilic and basophilic granulocyte as well as the monocyte, red cell and platelet lineages.

Animals↗
PubMed1988

RNA and protein analysis of an X-ray-induced hereditary cataract in rat.

We have analyzed the consequences of a mutation causing a recessive hereditary cataract in the rat. The lenses of these rats were examined both by histological and molecular biological methods. In the mutant rat lens development starts normally during early fetal life, but becomes progressively disrupted. The structure of the lens is clearly abnormal around the time of birth. Surprisingly, changes at the molecular level occur much later than the histological effects can be observed. Lens epithelial and cortex fiber cells apparently continue to synthesize the lens-specific crystallin mRNAs and proteins in spite of the damaged fiber cells.

Animals↗
PubMed1987

Differential expression of crystallin genes during development of the rat eye lens.

The concentrations of alpha A-, beta B1-, and total gamma-crystallin mRNAs were measured during development of the rat eye lens, using Northern blot and dot blot analysis. After 14 days of fetal growth a sharp increase in the concentration of all three mRNA species was observed. After birth, the concentration of alpha A-crystallin transcripts remains high until 6 months of age, the concentration of gamma-crystallin transcripts decreases gradually, while the concentration of beta B1-crystallin transcripts decreases sharply. The composition of the gamma-crystallin mRNA pool was determined by measuring the relative amount of the transcripts of each of the six gamma-crystallin genes using primer extension and S1-nuclease mapping. The transcripts of all six genes are found until the third month after birth. Thereafter the transcripts of the gamma 1-1, the gamma 3-1, and gamma 4-1 crystallin genes are no longer detectable. Later on the transcripts of the gamma 2-1 and gamma 2-2 genes also disappear leaving only the transcripts of the gamma 1-2 crystallin gene at the age of 8 months. The concentration of the six different gamma-crystallin mRNAs is thus regulated differentially.

Animals↗
PubMed1986

Sequence divergence and selection of cap sites in the rat gamma-crystallin gene family.

The transcription initiation sites of the six rat gamma-crystallin genes were mapped by combining the results of primer extension and S1 nuclease mapping experiments. To obtain more accurate results from the S1 nuclease mapping experiments, intron-deleted clones were constructed by a novel and efficient modification of existing methods involving the use of primer extension products to seal the exons. Four of the six gamma-crystallin genes have multiple transcription start sites. The major and most of the minor transcripts start with an adenosine. Analysis of the 5' flanking sequences of the gamma-crystallin genes shows that the sequence determining the position of the cap site is merely -CA- and that its optimal distance from the first T of the TATA box is 32 base pairs. Our data further suggest that an A to G transition in the first two base pairs of the Goldberg/Hogness box of one the genes does not affect the position of its major cap site. This, together with the fact that most minor transcription start sites are located upstream from the major cap sites, suggests that in the long TATA boxes of the rat gamma-crystallin genes the major RNA polymerase 'trap site' is not directly at the beginning of the TATA sequence.

Adenosine↗
PubMed1986

A vital staining method for measuring the efficiency of transfection of eucaryotic cells.

The xylE gene encodes catechol 2,3-dioxygenase, which catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde. The expression of this gene in eucaryotic cells can be detected simply by addition of catechol to the growth medium of the cells: cells that have a sufficient level of expression of the xylE gene stain yellow because of the accumulation of 2-hydroxymuconic semialdehyde. The number of stained cells is thus dependent upon the transfection efficiency as well as the level of expression of the xylE gene and is a measure of the combined transfection/expression efficiency in a particular cell type. Since the staining procedure does not affect the viability of the culture, the cells can be harvested afterward and analyzed for the expression of other, cotransfected, genes. This system for measuring transfection efficiency is especially useful when only small amounts of tissue are available.

Animals↗
PubMed1985

Characterization of the rat gamma-crystallin gene family and its expression in the eye lens.

Rat genomic clones, which together contain all of the rat genomic gamma-crystallin sequences, have been characterized. Five gamma-crystallin genes are located on a contiguous DNA region, 63 X 10(3) base-pairs long. These genes, named (5') gamma 1-1, gamma 1-2, gamma 2-2 and gamma 3-1 (3'), are all oriented head to tail. A sixth gamma-crystallin gene, named the gamma 4-1 gene, could not be linked to the gamma-crystallin gene cluster with our present set of genomic clones. Mapping experiments using single copy sequences which form the extreme 5' or 3' region of the gene cluster showed that, if the gamma 4-1 gene is located on the same chromosome, then it must be separated from the gene cluster by at least 25 X 10(3) base-pairs of DNA. All gamma-crystallin genes have a similar mosaic structure. They contain a large (0.9 X 10(3) to 1.88 X 10(3) base-pairs) intron in the middle of the gene and are further interrupted close to the 5' end of the gene. The length of the first exon varies from about 40 to about 50 base-pairs. The complementary DNA clone pRL-gamma-3 used in this study is a copy of the transcript of the gamma 3-1 gene, while the second complementary DNA clone, pRL-gamma-2, is most likely a copy of the transcript of the gamma 2-1 gene. It is further shown that rat lens messenger RNA protects fragments from the 3' ends of the four other gamma-crystallin genes against degradation by S1 nuclease, hence all six gamma-crystallin genes present in the rat genome must be transcribed in the lens. Repetitive sequences were found to be present between and around the gamma-crystallin genes. Mapping with cloned repetitive sequences showed that three different repeats, designated A, B and C, occur more than once in the gamma-crystallin gene cluster. Repeat C is also found in the gamma 4-1 region. A repetitive region 3' to the gamma 3-1 gene contains members of all three repeat families.

Animals↗