The role of integrins and extracellular matrix in anchorage-independent growth of a mammary carcinoma cell line

Anchorage-independent growth is a property of malignant cells. Extracellular matrix proteins are present in tumor spheroids but their function is not clearly defined. In this paper we show that a murine mammary carcinoma cell line, SP1, which expresses the fibronectin receptor alpha 5 beta 1 requires fibronectin for anchorage-independent growth in soft agar. Growth factors (hepatocyte growth factor and transforming growth factor-beta) also promote SP1 colony growth. In contrast, collagen types I and IV have an inhibitory effect on SP1 colony growth. A clone isolated from SP1 cells which expresses the collagen/laminin receptor alpha 2 beta 1 as well as the fibronectin receptor alpha 5 beta 1, demonstrates increased colony formation in the presence of fibronectin and collagen. These data suggest a role for both the alpha 5 beta 1 and alpha 2 beta 1 integrin receptors in the regulation of anchorage-independent growth of mammary carcinoma cells.     

Biochemical and functional characterization of recombinant von Willebrand factor produced on a large scale

Recombinant von Willebrand factor (r-vWF) was produced in serum-free medium on a large scale in recombinant Chinese hamster ovary cells and was purified from fermentation supernatant by a combination of anion exchange chromatography and heparin affinity chromatography. Heparin affinity chromatography yielded r-vWF polymers of different degrees of multimerization. r-vWF was analysed by qualitative and quantitative functional analysis. We could show that while binding of r-vWF to platelets did not depend on multimerization of the molecule, ristocetin-induced platelet aggregation, binding to collagen and binding to heparin correlated directly with the extent of multimerization. Binding of recombinant coagulation factor VIII (r-FVIII) to r-vWF was studied by real-time biospecific interaction analysis and surface plasmon technology. The data indicated that binding of r-FVIII did not depend on r-vWF multimerization. Real-time biospecific interaction analysis suggested a potential stoichiometry of 2 to 2.5 r-vWF subunits per r-FVIII molecule. Kinetic analysis of the r-vWF-r-FVIII interaction gave a binding rate constant of 3 x 10(6) M-1 s-1 and an association constant of 2.5 x 10(9) M-1. Reaction of r-vWF with carbohydrate-specific lectins demonstrated that r-vWF contained a high proportion of N-glycans composed of mannose, galactose, glucose, N-acetylglucosamine and terminal sialic acid. Carbohydrate moities were covalently bound to the protein structure and were quantitatively removed from r-vWF only after protein denaturation. The results demonstrated that r-vWF produced on large scale under serum-free culture conditions exhibited qualitative and quantitative functional properties comparable to human plasma-derived vWF.     

Functional domains of the alpha1 catalytic subunit of the AMP-activated protein kinase

The AMP-activated protein kinase is a heterotrimeric enzyme, important in cellular adaptation to the stress of nutrient starvation, hypoxia, increased ATP utilization, or heat shock. This mammalian enzyme is composed of a catalytic alpha subunit and noncatalytic beta and gamma subunits and is a member of a larger protein kinase family that includes the SNF1 kinase of Saccharomyces cerevisiae. In the present study, we have identified by truncation and site-directed mutagenesis several functional domains of the alpha1 catalytic subunit, which modulate its activity, subunit association, and protein turnover. C-terminal truncation of the 548-amino acid (aa) wild-type alpha1 protein to aa 312 or 392 abolishes the binding of the beta/gamma subunits and dramatically increases protein expression. The full-length wild-type alpha1 subunit is only minimally active in the absence of co-expressed beta/gamma, and alpha1(1-392) likewise has little activity. Further truncation to aa 312, however, is associated with a large increase in enzyme specific activity, thus revealing an autoinhibitory sequence between aa 313 and 392. alpha-1(1-312) still requires the phosphorylation of the activation loop Thr-172 for enzyme activity, yet is now independent of the allosteric activator, AMP. The increased levels of protein expression on transient transfection of either truncated alpha subunit cDNA are because of a decrease in enzyme turnover by pulse-chase analysis. Taken together, these data indicate that the alpha1 subunit of AMP-activated protein kinase contains several features that determine enzyme activity and stability. A constitutively active form of the kinase that does not require participation by the noncatalytic subunits provides a unique reagent for exploring the functions of AMP-activated protein kinase.     

Ocular and nasal irritation in operatives in Lancashire cotton and synthetic fibre mills

OBJECTIVES: To document the prevalence of work related ocular (eyeWRI) and nasal (noseWRI) irritation in workers in spinning mills of cotton and synthetic textile fibres and to relate the prevalence of symptoms to atopy, byssinotic symptoms, work history, and measured dust concentrations in the personal breathing zone and work area. METHODS: A cross sectional study of 1048 cotton workers and 404 synthetic fibre workers was performed. A respiratory questionnaire was given to 1452 workers (95% of the total available population). Atopy was judged by skin prick tests to three common allergens. Work area cotton dust sampling (WAdust) was carried out according to EH25 guidelines in nine of the 11 spinning mills included in the study. Personal breathing zone dust concentrations were assessed with the IOM sampler to derive total dust exposure (PTdust) and a concentration calculated after the removal of fly (Pless). RESULTS: 3.7% of all operatives complained of symptoms of byssinosis, 253 (17.5%) complained of eyeWRI and 165 (11%) of noseWRI. These symptoms did not relate to atopy or byssinosis, or correlate univariately with any measure of cotton dust exposure (noseWRI v WAdust r = 0.153, PTdust r = 0.118, eyeWRI v WAdust r = 0.029, PTdust r = 0.052). Both of these symptoms on logistic regression analysis were related to being of white origin (P < 0.001), female sex (P < 0.001), and younger age (P < 0.001). With regression analysis, there was a negative relation between dust concentration and prevalence of symptoms. CONCLUSION: Work related ocular and nasal irritation are the most common symptoms complained of by cotton textile workers. There was no relation between these symptoms and atopy, byssinosis, or dust concentration. It is likely that they relate to as yet unidentified agents unrelated to concentration of cotton dust.     

Methanethiosulfonate derivatives inhibit current through the ryanodine receptor/channel

To identify regions of the ryanodine receptor (RyR) important for ion conduction we modified the channel with sulfhydryl-reacting compounds. After addition of methanethiosulfonate (MTS) compounds channel conductance was decreased while other channel properties, including channel regulation by ATP, caffeine, or Ca, were unaffected. The site of action was accessible to the MTS compounds from the cytoplasmic, but not the luminal, side of the channel. In addition, the hydrophilic MTS compounds were only effective when the channel was open, suggesting that the compounds covalently modify the channel from within the water-filled ion conducting pathway. The decrease in channel current amplitude occurred in a step-wise fashion and was irreversible and cumulative over time, eventually leading to the complete block of channel current. However, the time required for each consecutive modification during continuous exposure to the MTS compounds increased, suggesting that successive modification by the MTS compounds is not independent. These results are consistent with the hypothesis that the channel forms a wide vestibule on the cytoplasmic side and contains a much smaller opening on the luminal side. Furthermore, our results indicate that the MTS compounds can serve as functional markers for specific residues of the RyR to be identified in molecular studies.