Richard F. Murphy

The regulatory role of TGF- β in airway remodeling in asthma

Both structural and inflammatory cells are capable of secreting transforming growth factor (TGF)‐β and expressing TGF‐β receptors. TGF‐β can induce multiple cellular responses including differentiation, apoptosis, survival and proliferation, and has been implicated in the development of several pathogenic conditions including cancer and asthma. Elevated levels of TGF‐β have been reported in the asthmatic airway. TGF‐β binds to its receptor complex and activates multiple pathways involving proteins such as Sma and Mad homologues, phosphatidylinositol‐3 kinase and the mitogen‐activated protein kinases, leading to the transcription of several genes. Cell type, cellular condition, and microenvironment, all play a role in determining which pathway is activated, which, in turn, is an indication of which gene is to be transcribed. TGF‐β has been shown to induce apoptosis in airway epithelial cells. A possible role for TGF‐β in the regulation of epithelial cell adhesion properties has also been reported. Enhancement of goblet cell proliferation by TGF‐β suggests a role in mucus hyper‐secretion. Elevated levels of TGF‐β correlate with subepithelial fibrosis. TGF‐β induces proliferation of fibroblast cells and their differentiation into myofibroblasts and extracellular matrix (ECM) protein synthesis during the development of subepithelial fibrosis. TGF‐β also induces proliferation and survival of and ECM secretion in airway smooth muscle cells (ASMCs), suggesting a possible cause of increased thickness of airway tissues. TGF‐β also induces the production and release of vascular endothelial cell growth factor and plasminogen activator inhibitor, contributing to the vascular remodeling in the asthmatic airway. Blocking TGF‐β activity inhibits epithelial shedding, mucus hyper‐secretion, angiogenesis, ASMC hypertrophy and hyperplasia in an asthmatic mouse model. Reduction of TGF‐β production and control of TGF‐β effects would be beneficial in the development of therapeutic intervention for airway remodeling in chronic asthma.

DNA methylation in breast and colorectal cancers

DNA methylation is one of several epigenetic changes observed in cells. Aberrant methylation of tumor suppressor genes, proto-oncogenes, and vital cell cycle genes has led many scientists to investigate the underlying cellular mechanisms of DNA methylation under normal and pathological conditions. Although DNA methylation is necessary for normal mammalian embryogenesis, both hypo- and hypermethylation of DNA are frequently observed in carcinogenesis and other pathological disorders. DNA hypermethylation silences the transcription of many tumor suppressor genes, resulting in immortalization of tumor cells. The reverse process, demethylation and restoration of normal functional expression of genes, is augmented by DNA methylation inhibitors. Recent studies suggest that DNA hypomethylation may also control gene expression and chromosomal stability. However, the roles of and relationship between hypomethylation and hypermethylation are not well understood. This review provides a brief overview of the mechanism of DNA methylation, its relationship to extrinsic stimulation including dietary intake and aging, and of abnormally methylated DNA in breast and colorectal cancers, which could be used as prognostic and diagnostic markers.

Significance of aromatic-backbone amide interactions in protein structure

Weakly polar interactions between aromatic rings of amino acids and hydrogens of backbone amides (Ar‐HN) have been shown to support local structures in proteins. Their role in secondary structures, however, has not been elucidated. To investigate the relationship between Ar‐HN interaction and the stability of local and secondary structures of polypeptides and to improve the prediction of this interaction based on amino acid sequence, the structures of 560 nonhomologous proteins, from the Protein Data Bank, were searched for Ar–HN interactions between the aromatic ring of each Phe, Tyr, and Trp residue at position i and the backbone amide group of any residue, except Pro, at the positions i, i − 1, i − 2, i − 3, i + 1, i + 2, and i + 3. Ar‐HN interactions were identified by calculating the chemical shift of the amide hydrogen caused by the proximal aromatic ring. Ar(i)–HN(i + 1, i + 2 and i + 3) interactions were more common (7.10%, 2.08%, and 0.54%, respectively) than were Ar(i)–HN(i − 1, i − 2, and i − 3) interactions (0.66%, <0.1%, and 0.18%, respectively). The value of the χ1 torsion angle of the aromatic residue in position i depended on the direction of the Ar–HN interaction. The position of the aromatic ring in Ar(i)–HN(i + 1, i + 2, and i + 3) interactions was mostly trans, in Ar(i)–HN(i − 1, i − 2, and i − 3) interactions mainly gauche(−), and in Ar(i)–HN(i) interactions mostly gauche(+). The analyses of the secondary structures of the protein fragments containing Ar–HN interactions showed that Ar–HN interactions were in all types of secondary structures. Search results suggest that Ar–HN interactions have a stabilizing effect on all types of secondary structures. Proteins 2001;43:373–381.

Pancreatic cancer cells require an EGF receptor-mediated autocrine pathway for proliferation in serum-free conditions

In-vitro and in-vivo studies have shown that autocrine growth factors and receptors are frequently expressed in human malignancies. Few of these studies, however, provide evidence that the identified autocrine pathway is functional. In this study, a functional autocrine growth pathway in pancreatic cancer has been identified using an in-vitro cell culture system. When pancreatic cancer cells were grown without change of medium, proliferation was greater than when either medium was replaced frequently (HPAF, CAPAN-2, PANC-1 or SW1990) or cells were grown in the presence of the EGF receptor tyrosine kinase inhibitor AG1478 or the MEK inhibitor PD098059 (HPAF or CAPAN-2). Activity of extracellular-regulated kinases (ERK) 1 and 2 and c- jun and c- fos mRNA levels were significantly elevated in CAPAN-2 cells cultured continuously in serum-free medium. Collectively, the observations indicate that the EGF receptor and the ERK MAP kinase pathway mediate autocrine signals. In contrast to previous reports, the GRP and IGF-I receptors were shown not to be required for autocrine effects on pancreatic cancer cell proliferation. Autocrine stimulation of the EGF receptor can contribute to sustained mitogenic activity and proliferation of pancreatic cancer cells.

Isolation of glucagon-like immunoreactivity of gut by affinity chromatography on anti-glucagon antibodies coupled to sepharose 4B

Abstract 1. 1. Antibodies raised in rabbits against porcine pancreatic glucagon were coupled to Sepharose 4B. The resultant complex was used to purify porcine pancreatic glucagon and glucagon-like immunoreactivity of pig ileum. The purified glucagon-like immunoreactivity was eluted from gel-filtration columns at positions corresponding to proteins of mol. wt. 12 000 (large glucagon-like immunoreactivity) and 3500 (small glucagon-like immunoreactivity). The former fraction accounted for over 95% of the total immunoreactivity. 2. 2. Large glucagon-like immunoreactivity contained at least three N-terminal amino acids, glycine, alanine and methionine, and was separated into three immunoreactive components by electrophoresis. 3. 3. Large glucagon-like immunoreactivity did not display the biological activity of pancreatic glucagon.

Nonlinear pharmacokinetics for the elimination of 5-fluorouracil after intravenous administration in cancer patients

SummaryPlasma concentrations of 5-fluorouracil (FU) and its primary catabolite, 5′, 6′-dihydro-5-fluorouracil (DHFU) were measured using gas-liquid chromatography after single-dose therapy with 7.2–14.4 mg/kg. Because of the limited sensitivity of the assay for drug levels in plasma, the urinary excretion of FU and metabolites was investigated using an ion-specific electrode after either a single bolus (7.0–9.6 mg/kg) or multiple-dose therapy (6.4–7.4 mg/kg/day). Half-life values for the elimination of FU from plasma (mean, 123.5 min) were greater in each patient than for the catabolite (mean, 109.2 min). Values of the area under the curve for FU profiles varied between patients (mean±SE, 12.7±1.9 μg·h/ml) by comparison with the relatively constant values for curves of DHFU concentrations (mean±SE, 2.8±0.15 μg·h/ml). In pharmacokinetic profiles of urinary excretion a transient phase of convex shape was apparent after 80%–98% of single doses of FU was excreted. Half-lives for the elimination of FU in urine were 2.6–5.9 h, which increased to 18–44 h on multiple dosing. The results demonstrate saturation in the elimination of FU after therapeutic doses, and are consistent with the proposal that reduction of FU to DHFU provides the rate-limiting step.

Following the TRAIL to apoptosis.

Apoptosis, programmed cell death, eliminates injured or harmful cells. It can mediate its response through the actions of death ligands including TRAIL. TRAIL, a member of TNF superfamily, induces apoptosis of transformed cells through the action of death domain receptors DR-4 and DR5. It directly induces apoptosis through an extrinsic pathway, which involes the activation of caspases. TRAIL also is able to prevent apoptosis through the actions of its decoy receptors DcR-1 and DcR-2. Various regulators of TRAIL include FADD, IAPs, Bcl-2s, p53, and FLIPs. TRAIL is present in cells involved in asthma including eosinophils, mast cells, fibroblasts, and airway epithelial cells. It is expressed in airway remodeling and may be linked with the pathways of transforming growth factor-beta1, which is thought to cause damage to the epithelium. The repair process of the epithelium is hindered as a result of increased apoptosis induced by TGF-β1, which overlaps with the pathways of TRAIL. Analogs of TRAIL could have therapeutical applications for asthma. TRAIL is also seen as the basis for a “miracle” drug for cancer because of its ability to selectively kill cancer cells.

Decoding IgE Fc receptors

Immunoglobulin E (IgE) plays a central role in the pathogenesis of allergic diseases by interacting with two membrane receptors: high-affinity FcεRI and low-affinity FcεRII (CD23). Allergen-induced IgE-occupied EcεRI aggregation on the mast cell or basophil cell surface leads to the activation of intracellular signaling events and eventually the release of pre-formed and de novo synthesized inflammatory mediators. The role of FcεRII in allergic diseases has been proposed to include regulation of IgE synthesis, enhanced histamine release from basophils, and a contribution to Ag-IgE complex presentation but the exact function of CD23 remains poorly understood. This review summarizes some new developments in IgE Fc-receptor studies with an emphasis on regulation of FcεRI expression and signal transduction, including monomeric IgE, lipid raft segregation, and some recently identified negative regulators. A better understanding of signaling events following IGE FcR aggregation will shed new light on how allergy patients might be treated more safely and effectively.

Synthesis of somatostatin by breast cancer cells and their inhibition by exogenous somatostatin and sandostatin

Three human breast cancer cell lines ZR-75-1, MDA-MB-436 and MCF-7 were found to contain respectively, 3.06, 2.69 and 1.86 fmol of somatostatin-like immunoreactivity (SLI) per 10(6) cells. Since SLI is undetectable in the passaging media it must, therefore, be synthesised by the cells. In the presence of fetal calf serum the cells were growth inhibited by addition of somatostatin or its long-lasting analogue, Sandostatin, but only after 3 days of continuous exposure. A 1-day exposure to either peptide had little or no effect on subsequent cell growth in peptide-free medium. Inhibition of cell proliferation is not due to cytotoxic effects of the dose used (500 ng ml-1, each) since both peptides caused short-term stimulation of growth in the absence of serum.

Enzymic iodination of the histidyl residue of secretin: a radioimmunoassay of the hormone.

Abstract Lactoperoxidase (EC 1.11.1.7) was a more efficient catalyst than chloramine-T for the incorporation of 125 I into secretin. Satisfactory radioimmunoassays were performed using the labelled product. Total hydrolysis and Edman degradation of the iodinated secretin showed that only the N-terminal histidyl residue of the hormone had been iodinated. Poly( l -histidine) was also iodinated using lactoperoxidase.