Absorption of protamine-insulin in diabetic patients. I. Preparation and characterization of protamine-125I-insulin.

Protamine-125I-insulin with low specific radioactivity was prepared using 125, 127I-insulin, 0.2 I/mole. The preparations were characterized by disc electrophoresis, isoelectric focusing and analytical gel chromatography in order to evaluate the suitability of 125I-insulin as marker for insulin in protamine-insulin. The stability of the preparations was followed up to 90 days at 4 degrees C. The biological and immunological activity was determined in mice, isolated rat fat cells and by radioimmunoassay. It was concluded that both from a chemical and a biological point of view, the protamine-125I-insulin is a satisfactory preparation that might be used in absorption studies.     

Stress-activated protein kinases are negatively regulated by cell density.

Stimulation by UV irradiation, TNFalpha, as well as PDGF or EGF activates the JNK/SAPK signalling pathway in mouse fibroblasts. This results in the phosphorylation of the N-terminal domain of c-Jun, increasing its transactivation potency. Using an antibody that specifically recognizes c-Jun phosphorylated at Ser63, we show that culture confluency drastically inhibited c-Jun N-terminal phosphorylation due to the inhibition of the JNK/SAPK pathway. Transfection experiments demonstrate that the inhibition occurs at the same level as, or upstream of, the small G-proteins cdc42 and Rac1. In contrast, the classical MAPK pathway was insensitive to confluency. The inhibition of JNK/SAPK activation depended on the integrity of the actin microfilament network. These results were confirmed and extended in monolayer wounding experiments. After PDGF, EGF or UV stimulation, c-Jun was predominantly phosphorylated in cells bordering the wound, which are the cells that move to occupy the wounded area. Thus, modulation of the stress-dependent signal cascade by confluency will restrict c-Jun N-terminal phosphorylation in response to mitogenic or chemotactic agents to cells that border a wounded area.