Search PubMed⌕ Search

Biomedical subjects

R L Kendall

Publications and source records attributed to R L Kendall.

12 recordsLinked to original sources
PubMed1999

Discovery of a small molecule insulin mimetic with antidiabetic activity in mice.

Insulin elicits a spectrum of biological responses by binding to its cell surface receptor. In a screen for small molecules that activate the human insulin receptor tyrosine kinase, a nonpeptidyl fungal metabolite (L-783,281) was identified that acted as an insulin mimetic in several biochemical and cellular assays. The compound was selective for insulin receptor versus insulin-like growth factor I (IGFI) receptor and other receptor tyrosine kinases. Oral administration of L-783,281 to two mouse models of diabetes resulted in significant lowering in blood glucose levels. These results demonstrate the feasibility of discovering novel insulin receptor activators that may lead to new therapies for diabetes.

Adenosine Triphosphate↗
PubMed1999

Vascular endothelial growth factor receptor KDR tyrosine kinase activity is increased by autophosphorylation of two activation loop tyrosine residues.

Vascular endothelial growth factor is an important physiological regulator of angiogenesis. The function of this endothelial cell selective growth factor is mediated by two homologous tyrosine kinase receptors, fms-like tyrosine kinase 1 (Flt-1) and kinase domain receptor (KDR). Although the functional consequence of vascular endothelial growth factor binding to the Flt-1 receptor is not fully understood, it is well established that mitogenic signaling is mediated by KDR. Upon sequencing several independent cDNA clones spanning the cytoplasmic region of human KDR, we identified and confirmed the identity of a functionally required valine at position 848 in the ATP binding site, rather than the previously reported glutamic acid residue, which corresponds to an inactive tyrosine kinase. The cytoplasmic domain of recombinant native KDR, expressed as a glutathione S-transferase fusion protein, can undergo autophosphorylation in the presence of ATP. In addition, the kinase activity can be substantially increased by autophosphorylation at physiologic ATP concentrations. Mutation analysis indicates that both tyrosine residues 1054 and 1059 are required for activation, which is a consequence of an increased affinity for both ATP and the peptide substrate and has no effect on kcat, the intrinsic catalytic activity of the enzyme. KDR kinase catalyzes phosphotransfer by formation of a ternary complex with ATP and the peptide substrate. We demonstrate that tyrosine kinase antagonists can preferentially inhibit either the unactivated or activated form of the enzyme.

DNA, Complementary↗
PubMed1998

Paracrine expression of a native soluble vascular endothelial growth factor receptor inhibits tumor growth, metastasis, and mortality rate.

Vascular endothelial growth factor (VEGF) is a potent and selective vascular endothelial cell mitogen and angiogenic factor. VEGF expression is elevated in a wide variety of solid tumors and is thought to support their growth by enhancing tumor neovascularization. To block VEGF-dependent angiogenesis, tumor cells were transfected with cDNA encoding the native soluble FLT-1 (sFLT-1) truncated VEGF receptor which can function both by sequestering VEGF and, in a dominant negative fashion, by forming inactive heterodimers with membrane-spanning VEGF receptors. Transient transfection of HT-1080 human fibrosarcoma cells with a gene encoding sFLT-1 significantly inhibited their implantation and growth in the lungs of nude mice following i.v. injection and their growth as nodules from cells injected s.c. High sFLT-1 expressing stably transfected HT-1080 clones grew even slower as s.c. tumors. Finally, survival was significantly prolonged in mice injected intracranially with human glioblastoma cells stably transfected with the sflt-1 gene. The ability of sFLT-1 protein to inhibit tumor growth is presumably attributable to its paracrine inhibition of tumor angiogenesis in vivo, since it did not affect tumor cell mitogenesis in vitro. These results not only support VEGF receptors as antiangiogenic targets but also demonstrate that sflt-1 gene therapy might be a feasible approach for inhibiting tumor angiogenesis and growth.

Animals↗
PubMed1996

Identification of a natural soluble form of the vascular endothelial growth factor receptor, FLT-1, and its heterodimerization with KDR.

A soluble form of the vascular endothelial growth factor (VEGF) receptor FLT-1 was identified in conditioned culture medium of human umbilical vein endothelial cells. The endogenous soluble FLT-1 (sFLT-1) receptor is chromatographically and immunologically similar to recombinant human sFLT-1 and binds [125I]VEGF with a comparable high affinity. Human sFLT-1 is shown to form a VEGF-stabilized complex with the extracellular domain of KDR in vitro, suggesting that not only full-length receptors are capable of forming ligand-induced heterodimeric complexes but also sFLT-1 can form a dominant negative complex with the mitogenically competent full-length KDR receptor.

Amino Acid Sequence↗
PubMed1995

Eukaryotic methionyl aminopeptidases: two classes of cobalt-dependent enzymes.

Using partial amino acid sequence data derived from porcine methionyl aminopeptidase (MetAP; methionine aminopeptidase, peptidase M; EC 3.4.11.18), a full-length clone of the homologous human enzyme has been obtained. The cDNA sequence contains 2569 nt with a single open reading frame corresponding to a protein of 478 amino acids. The C-terminal portion representing the catalytic domain shows limited identity with MetAP sequences from various prokaryotes and yeast, while the N terminus is rich in charged amino acids, including extended strings of basic and acidic residues. These highly polar stretches likely result in the spuriously high observed molecular mass (67 kDa). This cDNA sequence is highly similar to a rat protein, termed p67, which was identified as an inhibitor of phosphorylation of initiation factor eIF2 alpha and was previously predicted to be a metallopeptidase based on limited sequence homology. Model building established that human MetAP (p67) could be readily accommodated into the Escherichia coli MetAP structure and that the Co2+ ligands were fully preserved. However, human MetAP was found to be much more similar to a yeast open reading frame that differed markedly from the previously reported yeast MetAP. A similar partial sequence from Methanothermus fervidus suggests that this p67-like sequence is also found in prokaryotes. These findings suggest that there are two cobalt-dependent MetAP families, presently composed of the prokaryote and yeast sequences (and represented by the E. coli structure) (type I), on the one hand, and by human MetAP, the yeast open reading frame, and the partial prokaryotic sequence (type II), on the other.

Amino Acid Sequence↗
PubMed1994

Specificity of vascular endothelial cell growth factor receptor ligand binding domains.

Vascular endothelial cell growth factor binds with high affinity to FLT and KDR, two homologous tyrosine kinase receptors expressed on vascular endothelial cells. Placental growth factor, a vascular endothelial cell growth factor homologue, also binds with high affinity to the extracellular domains of FLT but not to the extracellular region of KDR. Vascular endothelial cell growth factor binds competitively with placental growth factor to the extracellular ligand binding domains of FLT, indicating that both ligands probably complex to overlapping or identical regions of this receptor.

Binding Sites↗
PubMed1993

Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor.

Vascular endothelial cell growth factor, a mitogen selective for vascular endothelial cells in vitro that promotes angiogenesis in vivo, functions through distinct membrane-spanning tyrosine kinase receptors. The cDNA encoding a soluble truncated form of one such receptor, fms-like tyrosine kinase receptor, has been cloned from a human vascular endothelial cell library. The mRNA coding region distinctive to this cDNA has been confirmed to be present in vascular endothelial cells. Soluble fms-like tyrosine kinase receptor mRNA, generated by alternative splicing of the same pre-mRNA used to produce the full-length membrane-spanning receptor, encodes the six N-terminal immunoglobulin-like extracellular ligand-binding domains but does not encode the last such domain, transmembrane-spanning region, and intracellular tyrosine kinase domains. The recombinant soluble human receptor binds vascular endothelial cell growth factor with high affinity and inhibits its mitogenic activity for vascular endothelial cells; thus this soluble receptor could act as an efficient specific antagonist of vascular endothelial cell growth factor in vivo.

Alternative Splicing↗
PubMed1992

Isolation and characterization of the methionine aminopeptidase from porcine liver responsible for the co-translational processing of proteins.

A methionine aminopeptidase that specifically removes methionine residues from peptides with amino-terminal sequences of Met-Ala-, Met-Val-, Met-Ser-, Met-Gly-, and Met-Pro- but not Met-Leu- or Met-Lys- has been isolated to homogeneity from porcine liver by a procedure involving five chromatographic steps. The enzyme, whose specificity matches that predicted for the entity responsible for the co-translational amino-terminal processing of nascent polypeptide chains, has a measured molecular mass of 70,000 Da by SDS-polyacrylamide electrophoresis and 67,000 Da by gel chromatography (under nondenaturing conditions), suggesting the native molecule is a monomer. It is activated by Co2+ and inhibited by beta-mercaptoethanol and EDTA. With octapeptide substrates related to the amino-terminal portion of the beta-chain of human hemoglobin (with a histidine in position 3), the enzyme had a pH optimum of 6.0. With a synthetic peptide devoid of histidine, it showed no pH dependence from 6.0 to 8.0. This sensitivity may be due to the propensity of peptides with histidine in the third position to bind divalent cations such as Co2+. The measured Km and kappa cat values were affected by residues in the second position. The peptide corresponding to the natural sequence (Met-Val-His-) gave a kappa cat/Km value of 260 mM-1 s-1; substitution of alanine in the second position raised the kappa cat/Km to 1523 mM-1 s-1, but substitution of proline lowered the value to 130. The effects are primarily on the kappa cat. The substitution of proline (for histidine) in the third position, the mutation found in hemoglobin Long Island, prevents the removal of the methionine residue, as occurs with the mutant protein. The porcine liver enzyme is similar to methionine aminopeptidases isolated from Escherichia coli, Salmonella typhimurium, and yeast in that it also is stimulated by Co2+. However, it is much larger than these enzymes and differs somewhat in specificity, particularly with the yeast enzyme.

Amino Acid Sequence↗
PubMed1991

Erythrocyte alloantigen loci Ea-D and Ea-I map to chromosome 1 in the chicken.

A test cross was conducted to analyse some linkage relationships in the chicken. Pea comb (P), naked neck (Na), tardy feathering (t), four erythrocyte alloantigen loci (Ea-C, -D, -I, -P), and the rearrangement break point (RB) of the NM 7092 t(Z;1) chromosome translocation were tested. Significant linkages were found between P and Ea-I (32.9 +/- 4.2), the RB and Ea-D (30.7 +/- 4.3), and t and Ea-D (38 +/- 4.8). The data suggest the linear order of t, Ea-D, and the RB, with t closest to the centromere. Significant linkage was also found between Na and Ea-P (32.4 +/- 4.9), confirming earlier reports.

Animals↗
PubMed1988

Expression of synthetic thaumatin genes in yeast.

Thaumatin is a plant protein that contains 8 disulfides and 207 amino acids in the mature form. The protein is of potential commercial interest since microgram quantities elicit an intense sweetness sensation. Two major variants of thaumatin have been identified in our laboratory by using sequence data obtained from thaumatin tryptic peptides. These differ by one amino acid at position 46 (asparagine or lysine), and both proteins differ from previously published sequences. We have synthesized DNA-coding sequences for three of these thaumatin variants using yeast preferred codons. The genes were inserted into an expression vector that contained a yeast 3-phosphoglycerate kinase promoter and terminator, and the vectors were transformed into yeast for expression of the recombinant protein. Upon lysis of the yeast cells, all thaumatin was localized in the insoluble cell fraction. Analysis of the sodium dodecyl sulfate solubilized yeast extracts by gel electrophoresis and Western blotting showed that thaumatin represented about 20% of the insoluble yeast protein. Although expressed at high levels, none of the thaumatins was biologically active (sweet). Preliminary protein folding experiments showed that two of three thaumatin variants could be folded to the sweet conformation.

Amino Acid Sequence↗