Regulation of the apoptotic threshold is of great importance in the homeostasis of both differentiating and fully developed organ systems. Triggering differentiation has been employed as a strategy to inhibit cell proliferation and accelerate apoptosis in malignant cells, in which the apoptotic threshold is often characteristically elevated. To better understand the mechanisms underlying differentiation-mediated regulation of apoptosis, we have studied death receptor responses during erythroid differentiation of K562 erythroleukemia cells, which normally are highly resistant to tumor necrosis factor (TNF) alpha-, FasL-, and TRAIL-induced apoptosis. However, upon hemin-mediated erythroid differentiation, K562 cells specifically lost their resistance to TNF-related apoptosis-inducing ligand (TRAIL), which efficiently killed the differentiating cells independently of mitochondrial apoptotic signaling. Concomitantly with the increased sensitivity, the expression of Programmed cell death or apoptosis is an active form of cellular destruction, which in concert with cell division controls immune responses, embryonic development, and tissue homeostasis. The most specific way to induce apoptosis is stimulation of the different types of death receptors, for example, tumor necrosis factor (TNF) receptor I (for review see reference 66), Fas/CD95 (28), or the TNF-related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5 (also known as TRAIL-R1 and -R2) (50,51,56). Stimulation of death receptors with their specific ligands FasL, TNF alpha (TNF-␣), and TRAIL causes in responsive cells oligomerization or aggregation of the receptor and activation of the cytoplasmic caspase machinery, resulting in disruption of normal cellular and nuclear morphology followed by DNA fragmentation and finally apoptosis (for review see reference 59).The TRAIL (67) receptors are members of the TNF receptor family and are widely expressed on the surface of many different cell types. The resistance of cells to TRAIL seems to be tightly regulated in a cell-type-and differentiation-dependent manner.

A bioluminescent Escherichia coli K-12 strain for the specific detection of the tetracycline group of antibiotics is described. A sensor plasmid, containing five genes from bacterial luciferase operon of Photorhabdus luminescens inserted under the control of tetracycline-responsive elements of the transposon Tn10, was constructed. Usage of the full-length luciferase operon in the sensor resulted in tetracycline-dependent light production without additions, i.e., self-luminescent phenotype, since all the substrates were intrinsically produced by the recombinant organism. The time needed for optimal induction of light emission was 90 min. Maximal induction of approximately 100-fold over uninduced levels by using 20 ng of tetracycline, and picomole sensitivities for the seven different tetracyclines tested, were obtained without added Mg2+ ions. The higher the pH and the magnesium ion concentration in the assay medium the higher was the amount of membrane-impermeable tetracycline-Mg2+ chelate complex. In consequence, by adjusting the pH and the Mg2+ ion concentration, the sensitivity of the assay can be modified for different analytical purposes. Different non-tetracycline antibiotics did not cause induction of light emission.

Collagenase-3 (MMP-13) is a matrix metalloproteinase capable of cleaving a multitude of extracellular matrix proteins in addition to fibrillar collagens. Human MMP-13 is expressed by fibroblasts in chronic cutaneous ulcers, but not in normally healing adult skin wounds. However, MMP-13 is produced by fibroblasts in adult gingival and in fetal skin wounds characterized by rapid collagen remodeling and scarless healing. Here, we have examined the role of human MMP-13 in remodeling of three-dimensional (3D) collagenous matrix by primary adult human skin fibroblasts. The high level of human MMP-13 expression by fibroblasts achieved by adenoviral gene delivery resulted in potent enhancement of remodeling and contraction of 3D collagen. Fibroblasts expressing MMP-13 in 3D collagen possessed altered filamentous actin morphology with patch-like actin distribution in cell extensions. The expression of MMP-13 promotes survival and proliferation of fibroblasts in floating collagen gel, and results in activation of Akt and extracellular signal-regulated kinase-1/2 by these cells. The results provide evidence for a novel role for human MMP-13 in regulating dermal fibroblast survival, proliferation, and interaction in 3D collagen, which may be an important survival mechanism for fibroblasts in chronic skin ulcers and contribute to scarless healing of adult gingival and fetal skin wounds.

We have developed assay technologies to measure hydrolyzing enzymes based on homogeneous time-resolved fluorescence quenching (TruPoint). High sensitivity was obtained using fluorescent europium chelates as labels, internally quenched by suitable quenchers and released upon enzymatic reaction. This approach allows robust and sensitive monitoring of low enzyme activities. The assay technology and the choice of donor-acceptor pairs were evaluated in three different enzymatic assays, a protease related to apoptosis, helicase involved in DNA unwinding, and phosphatase having an important role in cellular signaling cascades. All the assays produced an increasing signal, were sensitive, and had a good dynamic range. There were significant differences in optimized quenchers for each of the assays depending on the size, flexibility, and rigidity of the substrates. Also, clear differences in the energy-transfer reactions, their requirements for spectral overlapping, ionic interactions, and energy-transfer distances were found. Each of the enzymatic assays was briefly tested in a high-throughput screening environment by analyzing signal dynamics and statistical relevance as Z' factors.