mcl-1, a bcl-2 family member, was originally identified as an early gene induced during differentiation of ML-1 myeloid leukemia cells. In the present study, we demonstrate that Mcl-1 is tightly regulated by the granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling pathway. Upon deprivation of survival factor from TF-1 myeloid progenitor cells, Mcl-1 levels quickly dropped prior to visible detection of apoptosis of these cells. Upon restimulation of these deprived cells with GM-CSF, the mcl-1 mRNA was immediately induced and its protein product was accordingly resynthesized. Analysis with Ba/F3 cells expressing various truncation mutants of the GM-CSF receptor revealed that the membrane distal region between amino acids 573 and 755 of the receptor ␤ chain was required for mcl-1 induction. Transient-transfection assays with luciferase reporter genes driven by various regions of the mcl-1 promoter demonstrated that the upstream sequence between ؊197 and ؊69 is responsible for cytokine activation of the mcl-1 gene. Overexpression of mcl-1 delayed but did not completely prevent apoptosis of cells triggered by cytokine withdrawal. Its down regulation by antisense constructs overcame, at least partially, the survival activity of GM-CSF and induced the apoptosis of TF-1 cells. Taken together, these results suggest that mcl-1 is an immediate-early gene activated by the cytokine receptor signaling pathway and is one component of the GM-CSF viability response.

Molecular mechanisms underlying the repair of nitrosylated [Fe-S] clusters by the microbial protein YtfE remain poorly understood. The X-ray crystal structure of YtfE, in combination with EPR, magnetic circular dichroism (MCD), UV, and (17) O-labeling electron spin echo envelope modulation measurements, show that each iron of the oxo-bridged Fe(II) -Fe(III) diiron core is coordinatively unsaturated with each iron bound to two bridging carboxylates and two terminal histidines in addition to an oxo-bridge. Structural analysis reveals that there are two solvent-accessible tunnels, both of which converge to the diiron center and are critical for capturing substrates. The reactivity of the reduced-form Fe(II) -Fe(II) YtfE toward nitric oxide demonstrates that the prerequisite for N2 O production requires the two iron sites to be nitrosylated simultaneously. Specifically, the nitrosylation of the two iron sites prior to their reductive coupling to produce N2 O is cooperative. This result suggests that, in addition to any repair of iron centers (RIC) activity, YtfE acts as an NO-trapping scavenger to promote the NO to N2 O transformation under low NO flux, which precedes nitrosative stress.

We describe new experiments to measure the penetrative entrainment by a turbulent fountain in a steady two-layer stratification. A theoretical model was established by assuming that the stratification consists of two uniform layers, and the penetrative entrainment rate is estimated quantitatively by three independent formulae. Two quasi-uniform layers were observed in the steady state in the laboratory experiments. Experimental results gave a nearly constant dimensionless penetrative entrainment rate (0.65±0.17) across a density interface when the local Richardson number is smaller than 1.2.

Covalent attachment of poly(ethylene glycol) (PEG) molecules to drugs, proteins, and liposomes is a proven technology for improving their bioavailability, safety, and efficacy. Qualitative and quantitative analysis of PEG-derivatized molecules is important for both drug development and clinical applications. We previously reported the development of a monoclonal IgM antibody (AGP3) to PEG. We now describe a new IgG1 monoclonal antibody (E11) to PEG and show that it can be used in combination with AGP3 to detect and quantify PEG-derivatized molecules. Both antibodies bound the repeating subunits of the PEG backbone and could detect free PEG and PEG-modified proteins by ELISA, immunoblotting, and flow cytometry. Detection sensitivity increased with the length and the number of PEG chains on pegylated molecules. Both antibodies also efficiently accelerated the clearance of a PEG-modified enzyme in vivo. A sandwich ELISA in which E11/AGP3 were employed as the capture/detection antibodies was developed to detect PEG-modified proteins at concentrations as low as 1.2 ng/mL. In addition, the ELISA could also quantify, in the presence of 10% fetal bovine serum, free methoxy-PEG20,000, PEG2,000-quantum dots, and PEG2,000-liposomes at concentrations as low as 20 ng/mL (1.0 nM), 1.4 ng/mL (3.1 pM), and 2.4 ng/mL (3.13 nM phospholipids), respectively. Finally, we show that the sandwich ELISA could accurately measured the in vivo half-life of a PEG-modified enzyme. These antibodies should be generally applicable to the qualitative and quantitative analysis of all PEG-derivatized molecules.