Delineation of the IGF-II C domain elements involved in binding and activation of the IR-A, IR-B and IGF-IR.

OBJECTIVE: Human insulin-like growth factor-I and -II (IGF-I and -II) ligands share a high degree of sequence and structural homology. Despite their similarities, IGF-I and IGF-II exhibit differential receptor binding and activation characteristics. The C domains of IGF-I and IGF-II are the primary determinants of binding specificity to the insulin-like growth factor I receptor (IGF-IR), insulin receptor exon 11- (IR-A) and exon 11+ (IR-B) isoforms. DESIGN: Three IGF-II analogues were generated in order to delineate the C domain residues that confer the differential receptor binding affinity and activation properties of the IGFs. Chimeric IGF-II analogues IGF-IICI(N) and IGF-IICI(C) contained partial IGF-I C domain substitutions (IGF-I residues underlined) GYGSSSRRSR and SRVSRRAPQT, respectively. RESULTS: The IGF-IICI(N) analogue bound the IR-A and IGF-IR with high affinity but bound the IR-B with a relatively lower affinity than IGF-II, suggesting a negative interaction between the exon-11 encoded peptide in the IR-B and the C-domain. The ability of IGF-IICI(N) to activate receptors and elicit cell viability responses was generally proportional to its relative receptor binding affinity but appeared to act as a partial agonist equivalent to IGF-I when binding and activating the IGF-IR. In contrast, IGF-IICI(C) bound IGF-IR with high affinity but elicited lower receptor activation and cell viability responses. Analogue IGF-IICI(S) contained a truncated IGF-I C domain (GSSSRRAT) and generally displayed a relatively poor ability to bind, activate and elicit viability responses via each receptor. CONCLUSIONS: Together, the IGF analogues demonstrate that both flanks of the IGF-II C domain play important roles in the greater ability of IGF-II to bind and activate IR receptors than IGF-I.

Serum concentrations of brain-derived neurotrophic factor (BDNF) are decreased in colorectal cancer patients.

OBJECTIVE: To determine the usefulness of brain-derived neurotrophic factor (BDNF) as a diagnostic biomarker for colorectal cancer (CRC). MATERIALS AND METHODS: ELISA immunoassay was used to examine BDNF concentrations in the sera of two different retrospective cohorts consisting of CRC patients and age/gender matched controls. Cohort 1 consisted of 99 controls and 97 CRC patients, whereas cohort 2 consisted of 47 controls and 91 CRC patients. RESULTS: In cohort 1, the median concentration of BDNF was significantly (p< 0.0001) lower in CRC patient samples (18.8 ng/mL, range 4.0-56.5 ng/mL) than control samples (23.4 ng/mL, range 3.0-43.1 ng/mL). This finding was validated in an independent patient cohort (CRC patients: 23.0 ng/mL, range 6.0-45.9 ng/mL; control patients: 32.3 ng/mL, range 14.2-62.4 ng/mL). BDNF concentrations did not differ significantly between Dukes' staging in the patient cohort, however patients with Stages A, B, C and D (p< 0.01 for each stage) tumours had significantly reduced BDNF levels compared to healthy controls. Receiver operating characteristic analysis was performed to determine the ability of BDNF to discriminate between healthy controls and those with CRC. At 95% specificity, BDNF concentrations distinguished CRC patients with 25% and 18% sensitivity, respectively, in cohorts 1 and 2 (cohort 1: AUC=0.79, 95% CI 0.70-0.87; cohort 2: AUC =0.69, 95% CI 0.61-0.76). CONCLUSION: The serum levels of BDNF were significantly lower in colorectal cancer patients when compared to a control population, and this did not differ between different Dukes' stages.

Purification and molecular characterization of recombinant rat betacellulin.

A method for the large scale expression and purification of rat betacellulin (BTC) from Escherichia coli has been developed using a cleavable fusion protein strategy. Insoluble fusion protein collected as inclusion bodies was dissolved in urea under reducing conditions, re-folded, and purified by gel filtration chromatography and C(4) RP-HPLC. Authentic rat BTC was obtained after proteolytic cleavage of the fusion protein with Factor Xa. Factor Xa cleaved an additional site within the BTC protein, generating a truncated isoform separable from full-length BTC by heparin-affinity chromatography. Recombinant rat BTC stimulated the proliferation of mouse Balb/c 3T3 fibroblasts and competed for binding to the ErbB1 receptor in a dose-dependent manner analogous to that of BTC purified from natural sources.

Measurement of betacellulin levels in bovine serum, colostrum and milk.

Betacellulin, a member of the epidermal growth factor (EGF) family, was originally isolated and identified from the conditioned medium from a murine pancreatic beta-cell carcinoma cell line. Recently, we isolated bovine betacellulin from a growth factor enriched cheese whey extract, but there is no information on the presence of betacellulin in other biological fluids. We have cloned the cDNA for bovine betacellulin, produced recombinant betacellulin and shown that it has a similar potency to the purified native molecule in stimulating the proliferation of Balb/c3T3 fibroblasts. We have produced a polyclonal antiserum to bovine betacellulin which did not cross-react with EGF or transforming growth factor-alpha (TGF-alpha). The antibody was used in a homologous RIA that was able to detect betacellulin in pooled bovine colostrum sampled during the first 3 days after calving (2.30+/-0.11 ng/ml mean+/-s.e.m.; n=6), in bovine milk soluble fraction (1.93+/-0.64 ng/ml mean+/-s.e.m.; n=5) and in bovine cheese whey (2.59+/-0.16 ng/ml mean+/-s.e.m.; n=3). The betacellulin concentration in foetal bovine serum (FBS) (3.68+/-0.59 ng/ml mean+/-s.e.m.; n=6) greatly exceeded that of betacellulin in serum from male calves 1 and 5 weeks of age (0.53+/-0.15 ng/ml and 0.70+/- 0.09 ng/ml respectively; mean+/-s.e.m.; n=9). Betacellulin measured in the serum of these same animals when aged between 27 and 43 weeks was below the detection limits of the RIA. Sera from 10 out of 36 unmated heifers contained betacellulin levels within the detection limits of the assay (0.433+/-0.06 ng/ml mean+/-s.e.m.; n=10). The presence of betacellulin in bovine colostrum and milk suggests that it plays a role in the growth and development of the neonate and/or mammary gland function. The results also show that betacellulin is undetectable in the castrated adult male circulation. Additionally, although present in very low amounts, serum betacellulin could be under hormonal regulation in the female, since betacellulin was detected in sera from 27% of the unmated heifers examined in this study. The high levels of betacellulin detected in FBS relative to newborn and adult serum suggests a possible endocrine role for this growth factor in the bovine foetus.

Identification of betacellulin as a major peptide growth factor in milk: purification, characterization and molecular cloning of bovine betacellulin.

Betacellulin (BTC), a member of the epidermal growth factor (EGF) family of peptide growth factors, was purified from a growth-factor-enriched whey fraction of bovine milk by a combination of ion-exchange chromatography, gel-filtration chromatography, affinity chromatography and reverse-phase HPLC. Bovine BTC (bBTC) had an apparent molecular mass of 21-22 kDa on SDS/PAGE and exists in a glycosylated form. The cDNA encoding bBTC was obtained by a combination of 5' and 3' rapid amplification of cDNA ends ('RACE'). The primary translation product consists of 178 amino acid residues containing a putative signal sequence, a transmembrane domain, the mature BTC domain and a cytoplasmic domain containing a highly hydrophilic Arg-Lys-rich region similar to that of mouse BTC and human BTC. The amino acid sequence of the bBTC precursor was 88% identical with human BTC and 79% identical with mouse BTC. The bBTC gene was found to be expressed in a wide range of tissues, including the mammary gland. The identification of BTC in milk raises the possibility that it has a major role in the growth and development of the neonatal gastrointestinal tract.

Milk-derived growth factors as serum supplements for the growth of fibroblast and epithelial cells.

We have investigated the response of several epithelial and fibroblastic cells to a mitogenic extract of bovine milk. Cation exchange chromatography was used to produce a mitogen-rich fraction from an industrial whey source that, although comprising only 0.5% of total whey protein, contained the bulk of the growth factor activity. This fraction was a source of potent growth promoting activity for all mesodermal-derived cells tested, including human skin and embryonic lung fibroblasts, Balb/c 3T3 fibroblasts, and rat L6 myoblasts. Maximal growth of all these cell types exceeded that observed in 10% fetal bovine serum. Feline kidney and baby hamster fibroblasts and Chinese hamster ovary cells were less responsive, achieving a maximal growth response of 50-75% that observed in 10% fetal bovine serum. Maximal growth achieved in whey-extract-supplemented cultures of Balb/c 3T3 and human skin fibroblasts, and L6 myoblast cultures exceeded that seen in response to recombinant acidic or basic fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor, or epidermal growth factor. Importantly, addition of low concentrations of fetal bovine serum to the whey-derived mitogenic fraction produced an additive response. However, concentrated milk-derived factors were found to be inhibitory to the growth of all epithelial lines tested, including rat intestinal epithelial cells, canine kidney epithelial cells, and mink lung cells. It is concluded that industrial whey extracted in this form constitutes an important source of potent growth-promoting agents for the supplementation of mesodermal-derived cell cultures.