The angiogenic receptor KDR is widely distributed in human tissues and tumours and relocates intracellularly on phosphorylation. An immunohistochemical study.

AIMS: Angiogenesis is an important factor in tumour growth and metastasis. Vascular endothelial growth factor receptor 2 (VEGFR-2) or KDR plays a crucial role in angiogenesis. The aim of this study was to raise and characterize antibodies against phosphorylated KDR which could be used for studies on human tissues to assess KDR activation and novel inhibitors of KDR activation in clinical trials. METHODS AND RESULTS: Three monoclonal antibodies and one rabbit polyclonal antiserum were produced. The specificity of the antibodies was confirmed by ELISA. One of the mouse antibodies and the rabbit polyclonal antiserum reacted with a 200-kDa band on a Western blot of human umbilical vein endothelial cell (HUVEC) lysates, the molecular weight of KDR. Immunohistochemical staining showed that phosphorylated KDR is present in a wide variety of normal tissues including liver, colon and placenta, and is not restricted to endothelium. It was also present in a number of human tumours including breast carcinomas, colonic carcinomas and non-Hodgkin's lymphomas. The pattern of staining was membranous, cytoplasmic and nuclear. CONCLUSIONS: This study has shown that phosphorylated KDR is present in a wide variety of tumour and tissue types and is not confined to endothelium.

McTimoney chiropractic: a gentle way with back pain.

There are 1080 chiropractors in the UK today, and almost one-third of these are McTimoney chiropractors. This article outlines the development of McTimoney Chiropractic, which is a particular branch of the chiropractic profession in the UK, taught at the McTimoney Chiropractic College in Abingdon, near Oxford. The McTimoney method is distinguished by its gentle, whole body approach. It aims to correct the alignment of the bones of the spine and other joints of the body, to restore nerve function, to alleviate pain, and to promote natural health. The technique is suitable for the very young as well as the old and frail. In this paper, several nurses-turned-chiropractor offer their personal views. Reference is also made to the McTimoney-Corley technique, which is a similar chiropractic method taught at the Oxford School of Chiropractic, UK.

Structure of a specific peptide complex of the carboxy-terminal SH2 domain from the p85 alpha subunit of phosphatidylinositol 3-kinase.

We have determined the solution structure of the C-terminal SH2 domain of the p85 alpha subunit of human phosphatidylinositol (PI) 3-kinase (EC 2.7.1.137) in complex with a phosphorylated tyrosine pentapeptide sequence from the platelet-derived growth factor receptor using heteronuclear nuclear magnetic resonance spectroscopy. Overall, the structure is similar to other SH2 domain complexes, but displays different detail interactions within the phosphotyrosine binding site and in the recognition site for the +3 methionine residue of the peptide, the side chain of which inserts into a particularly deep and narrow pocket which is displaced relative to that of other SH2 domains. The contacts made within this +3 pocket provide the structural basis for the strong selection for methionine at this position which characterizes the SH2 domains of PI3-kinase. Comparison with spectral and structural features of the uncomplexed domain shows that the long BG loop becomes less mobile in the presence of the bound peptide. In contrast, extreme resonance broadening encountered for most residues in the beta D', beta E and beta F strands and associated connecting loops of the domain in the absence of peptide persists in the complex, implying conformational averaging in this part of the molecule on a microsecond-to-millisecond time scale.

Specific inhibition of epidermal growth factor receptor tyrosine kinase by 4-anilinoquinazolines.

Since the mitogenic action of EGF is mediated by ligand-induced autophosphorylation of the EGF receptor (EGFR), and EGFR is commonly overexpressed in solid human tumours, inhibitors of receptor tyrosine kinase activity (RTK) could prove to be effective antitumour agents. Screening of a compound library using an EGF-RTK enzyme prepared from human tumour derived A431 cells identified a series of potent (IC50 < 1 microM) enzyme inhibitors. These inhibitors are quinazolines bearing a variety of substituted anilines at the 4-position. The most potent 4-anilinoquinazolines (IC50 approximately equal to 20 nM) have small non-polar meta substituents on the aniline ring, and are competitive with ATP and non-competitive with substrate. The growth inhibitory activity of these agents was assessed in vitro using KB cells (human oral squamous tumour) grown in the absence or presence of EGF. A selected compound, 4-(3-chloroanilino)quinazoline (CAQ), inhibited EGF-stimulated growth in a concentration dependent manner and complete blockade was observed at concentrations (1-10 microM) which had no effect on basal growth. Selectivity of growth inhibition by CAQ was further exemplified in IGF1-stimulated KB cells where no effect was detected at concentrations which completely blocked EGF-stimulated growth. Similarly, CAQ blocked TGF alpha-stimulated growth in MCF-7 human breast cancer cells without affecting insulin-stimulated growth. These studies define a novel class of EGF-RTK inhibitors which are also potent and selective inhibitors of EGF-stimulated human tumour cell growth in vitro.

Transforming growth factor alpha and epidermal growth factor levels in normal human gastrointestinal mucosa.

Acid soluble proteins from 23 samples of normal human gastrointestinal mucosa derived from four normal adult organ donors were extracted and subjected to specific radiommunoassays for transforming growth factor alpha (TGF alpha) and urogastrone epidermal growth factor (URO-EGF). All tissues were found to contain immunoreactive TGF alpha and levels ranged from 57 to 4,776 pg-1 wet weight of tissue. Although levels varied between tissue donors, the distribution of TGF alpha throughout the gastrointestinal tract appeared similar in all cases. URO-EGF levels were much lower (0-216 pg g-1 wet weight). TGF alpha levels in extracts of gastrointestinal mucosa from a 7-year-old female donor were higher and the observed distribution was markedly different from adult levels. URO-EGF was not detected in mucosal or submucosal tissue extracts from this patient. Further studies in juveniles are indicated.