Fusion of the CH1 domain of IgG1 to epidermal growth factor (EGF) prolongs its retention in the blood but does not increase tumor uptake

An expression vector (pJW4) for a human epidermal growth factor (hEGF)-CH1 fusion protein was constructed by fusing the gene for hEGF with the gene for CH1 of murine IgG1 with/without a peptide linker sequence [(GGGGS)3] and inserting the recombinant gene into vector pGEX2T. Expression vector pGEX2T was transfected into E. coli (BL-21) and hEGF-CH1 expressed by induction of the lac Iq promotor with 50 microM isopropyl beta-D-thiogalactopyranoside (IPTG). hEGF- CH1 fused to glutathione S-transferase (GST) was isolated and purified by affinity chromatography. GST was cleaved using thrombin. SDS-PAGE demonstrated a protein with the expected M(r) (18 kDa) positive for hEGF by Western blot. hEGF-linker-CH1 exhibited preserved binding to A431 (2-3 x 10(6) EGFR/cell) and MDA-MB-468 breast cancer cells (1-2 x 10(6) EGFR/cell). hEGF-CH1 without the linker exhibited poor receptor binding. hEGF-linker-CH1 also exhibited strong binding to soluble EGFR equivalent to that of hEGF. The tumor and normal tissue distribution of hEGF-linker-CH1 labeled with 123I was compared with 123 I-hEGF at 24 h after i.v. injection to mice implanted with s.c. MDA-MB-468 xenografts. Fusion of hEGF with CH1 increased its retention in the blood 14-fold but did not significantly increase tumor uptake. Tumor/blood ratios were higher for hEGF than for hEGF-linker-CH1. We conclude that hEGF is more attractive than hEGF-linker-CH1 for imaging EGFR-positive tumors.     

Insulin,insulin-like growth factor-I (IGF-I), IGF binding proteins,their biologic interactions,and colorectal cancer

Secular changes and worldwide variations in incidence rates of colorectal cancer, along with results from twin and migrant studies, provide compelling evidence that environmental factors influence the risk of this disease. Among the most important of these factors are diet and associated factors, such as physical activity and body size. Recent data suggest that dietary and related factors may influence colorectal cancer risk via their effects on serum insulin concentrations and on the bioavailability of insulin-like growth factor-I (IGF-I). Epidemiologic studies have shown that IGF-I is positively associated with the risk of colorectal cancer, and experimental studies have shown that IGF-I has mitogenic and antiapoptotic actions on colorectal cancer cells. IGF-I bioactivity is regulated in part by its six binding proteins (IGFBP-1 to IGFBP-6); insulin inhibits the production of IGFBP-1 and perhaps IGFBP-2. As a result, chronically elevated fasting and postprandial insulin levels may lead to a decrease in circulating IGFBP-1 and IGFBP-2 concentrations and, consequently, an increase in IGF-I bioavailability. Insulin may also increase the circulating IGF-I/IGFBP-3 ratio by increasing hepatic growth hormone sensitivity. The increased IGF-I bioavailability may, over time, increase the risk of colorectal cancer. This new evidence for biologic interactions among insulin, IGF-I, and IGFBPs in the context of colorectal carcinogenesis provides a potential mechanism through which diet and associated factors may increase the risk of this cancer.     

Opioid regulation of gonadotropin release: role of signal transduction cascade

The present investigation elucidates the opioidergic modulation of gonadotropin releasing hormone release mechanism by signal transduction cascade in discrete brain regions from estrogen-progesterone primed ovariectomized rats. The effects of mu-opioid agonist morphine and its antagonist naloxone followed by morphine were studied (in two different groups of rats) on protein kinase A, adenosine 3',5' cyclic monophosphate, protein kinase C and calcium/calmodulin protein kinase-II as well as phospholipase C, phospholipase A(2), diacylglycerol and inositol 1,4, 5-triphosphate. Significant decline in phosphoinositide metabolism was observed after morphine treatment as depicted by decrease in phospholipase C and phospholipase A2 activities as well as inositol 1,4,5-triphosphate and diacylglycerol contents from discrete brain regions. Protein kinase A activity showed translocation from membrane bound to cytosolic form along with a decrease in its activator adenosine 3',5'-cyclic monophosphate levels in morphine-treated group. Calcium/calmodulin dependent protein kinase II activity also declined, whereas, protein kinase C activity increased in the cytosolic fraction after 45 min of morphine administration. Naloxone was seen to counteract the changes induced by morphine in most of the brain regions studied.Morphine also suppressed luteinizing hormone levels, whereas, follicle stimulating hormone level did not change. The present investigation provides evidence for opioidergic mediated suppression of gonadotropin release through the downregulation of signal transduction cascade.     

Inhibition of the contractile responses of isolated human and rat bladders by clenbuterol

PURPOSE: We investigated the inhibition of the contractile responses of human continent and unstable detrusor muscle by the beta2 agonist clenbuterol as well as the inhibition of electrical field stimulation evoked contractile responses of isolated rat bladder muscle strips by orally administered clenbuterol. MATERIALS AND METHODS: The contractile responses of human continent and unstable detrusor muscle strips to electrical field stimulation (0.05 milliseconds, 0.5 to 80 Hz.) were measured before and after adding 10(-9) to 10(-4) M. clenbuterol in vitro. In addition, 6 rats per group were dosed orally with 2 microg x kg(-1) clenbuterol daily acutely (1 dose) or chronically (1 dose daily for 8 days), or with distilled water to serve as controls. The contractile response to electrical field stimulation of strips of isolated detrusor muscle was then measured. Serum clenbuterol levels were analyzed in duplicate by enzyme-linked immunosorbent assay and high performance liquid chromatography. RESULTS: In vitro clenbuterol significantly inhibited the electrical field stimulation evoked contractile responses of detrusor muscle strips from unstable but not continent human bladders. A significant inhibitory effect of clenbuterol on the electrical field stimulation evoked contractile response of rat detrusor muscle was observed after chronic but not acute oral dosing (p <0.01). Serum clenbuterol levels measured by enzyme-linked immunosorbent assay and high performance liquid chromatography were not significantly different. CONCLUSIONS: Clenbuterol or related beta2-adrenoceptor agonists may represent a useful therapeutic strategy for detrusor muscle overactivity.     

Evaluation of 10 chemicals for aneuploidy induction in the hexaploid wheat assay.

This study was a part of an international project sponsored by the Commission of the European Communities to evaluate the utility of certain bioassays including hexaploid wheat assay to identify potential aneugens. Ten suspect spindle poisons, i.e. colchicine (COL), cadmium chloride (CdCl2), chloral hydrate (CH), diazepam (DIZ), econazole (EZ), hydroquinone (HQ), pyrimethamine (PY), thiabendazole (TB), thimerosal (TM), and vinblastin sulphate (VBL) were tested for their ability to induce green and/or white leaf sectors as indicators of loss or gain of a chromosome respectively, in Neatby's strain of Chinese Spring wheat (2n = 6x = 42). All the chemicals tested in this study, with the exception of CH and HQ yielded positive response.     

In vivo monastral blue-induced lamellar-bodies in lysosomes of pulmonary intravascular macrophages (PIMs) of bovine lung: Implications of the surface coat

We previously reported that the pulmonary intravascular macrophages (PIMs) of sheep, goat, and calf lung contained a heparin and a lipolytic lipase sensitive surface coat by using tannic acid as a component of paraformaldehyde-glutaraldehyde-based fixative. The implication of this sensitivity was that the surface coat was predominantly comprised of lipoprotein-like substance. In this study we report that monastral blue (MB) used as a vascular tracer interacted with the coat globules and lost its original particulate appearance. Its precise localization in the PIMs was in combination with altered macromolecules of the surface coat in the form of lipid droplets, which conformed to the conventional view of neutral lipids. In contrast, pigment particles examined in their native state resembled metalic particles as electron-dense eliptical rods. The lipid droplets were subsequently internalized through endocytic route and found their access into the lysosomal compartments of PIMs at the electron microscopic level. Lamellar bodies (LLBs) arose from the lysosomal matrix after the entry of lipid droplets in the secondary lysosomes. Acid phosphatase activity was located in secondary lysosomes as well as in endosomes. These observations suggest that coat granules of the PIMs acted as a carrier of exogenous MB particles to deliver the complex to the lysosomal compartment where partial digestion lead to the formation of lamellar bodies. The implications of MB (cationic dye) as a vascular tracer for studying phagocytic index of PIMs in the light of their coat and the rapid development of LLBs are discussed. It is proposed that MB by initially combining with the surface coat provokes mobilization of intracellular lipid pools. In this way metabolism of vasoactive lipid in the PIMs is stimulated to influence the dynamics of pulmonary circulation in the calves.© Willey-Liss, Inc.