During their lifespan, immature cells normally pass through sequential transitions to a differentiated state and eventually undergo cell death. This progression is aberrant in cancer, although the transition to differentiation can be reestablished in inducible leukemia cell lines. This report describes a gene, MCLl, that we isolated from the ML-1 human myeloid leukemia cell line during phorbol ester-induced differentiation along the monocyte/macrophage pathway. Our results demonstrate that expression ofMCLI increases early in the induction, or "programming," of differentiation in ML-1 (at 1-3 hr), before the appearance of differentiation markers and mature morphology (at 1-3 days). They further show that MCLI has sequence similarity to BCL2, a gene involved in normal lymphoid development and in lymphomas with the t(l4;18) chromosome translocation. MCLI and BCL2 do not fall into previously known gene families. BCL2 differs from many oncogenes in that it inhibits programmed cell death, promoting viability rather than proliferation; this parallels the association of MCL1 with the programming of differentiation and concomitant maintenance of viability but not proliferation. Thus, in contrast to proliferation-associated genes, expression of MCLI and BCL2 relates to the programming of differentiation and cell viability/death. The discovery of MCLI broadens our perspective on an emerging MCLI/BCL2 gene family and will allow further comparison with oncogene families.

Norbinaltorphimine (NorBNI), guanidinonaltrindole, and atrans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl) piperidine (JDTic) are selective opioid receptor (KOR) antagonists having very long durations of action in vivo despite binding non-covalently in vitro and having only moderately high affinities. Consistent with this, we found that antagonist treatment significantly reduced the subsequent analgesic response of mice to the KOR agonist U50,488 in the tail-withdrawal assay for 14 -21 days. Receptor protection assays were designed to distinguish between possible explanations for this anomalous effect, and we found that mice pretreated with the readily reversible opioid antagonists naloxone or buprenorphine before norBNI responded strongly in the tail-flick analgesia assay to a subsequent challenge with U50,488 1 week later. Protection by a rapidly cleared reagent indicates that norBNI did not persist at the site of action. In vitro binding of [

Smart cyanine-grafted gadolinium oxide nanocrystals (Cy-GdNCs) obtained by albumin-based biomineralization are shown to be theranostic nanocomposites, with promising properties for trimodal near-infrared fluorescence/photoacoustics/magnetic-resonance imaging-guided photothermal tumor ablation.

Macrophages acquire distinct phenotypes during tissue stress and inflammatory responses, but the mechanisms that regulate the macrophage polarization are poorly defined. Here we show that tuberous sclerosis complex 1 (TSC1) is a critical regulator of M1 and M2 phenotypes of macrophages. Mice with myeloid-specific deletion of TSC1 exhibit enhanced M1 response and spontaneously develop M1-related inflammatory disorders. However, TSC1-deficient mice are highly resistant to M2-polarized allergic asthma. Inhibition of the mammalian target of rapamycin (mTOR) fails to reverse the hypersensitive M1 response of TSC1-deficient macrophages, but efficiently rescues the defective M2 polarization. Deletion of mTOR also fails to reverse the enhanced inflammatory response of TSC1-deficient macrophages. Molecular studies indicate that TSC1 inhibits M1 polarization by suppressing the Ras GTPase-Raf1-MEK-ERK pathway in mTOR-independent manner, whereas TSC1 promotes M2 properties by mTOR-dependent CCAAT/enhancer-binding protein-b pathways. Overall, these findings define a key role for TSC1 in orchestrating macrophage polarization via mTOR-dependent and independent pathways.

AgS nanoparticles are increasingly important in biomedicine, such as in cancer imaging. However, there has been only limited success in the exploration of theranostic AgS nanoparticles for photoinduced cancer imaging and simultaneous therapy. Here we report size-dependent AgS nanodots (NDs) with well-defined nanostructure as a theranostic agent for multimodal imaging and simultaneous photothermal therapy. The NDs are precisely synthesized through carefully controlled growth of AgS in hollow human serum albumin nanocages. These NDs produce effective fluorescence in second near-infrared (NIR-II) region, distinct photoacoustic intensity, and good photothermal conversion in a size-dependent manner under light irradiation, thereby generating sufficient in vivo fluorescence and photoacoustic signals as well as potent hyperthermia at tumors. Moreover, AgS NDs possess ideal resistance to photobleaching, effective cellular uptake, preferable tumor accumulation, and in vivo elimination, thus facilitating NIR-II fluorescence/photoacoustics imaging with both ultrasensitivity and microscopic spatial resolution and simultaneous photothermal tumor ablation. These findings provide insight into the clinical potential of AgS nanodots for cancer theranostics.

Abstract.A family of genes related to the bcl-2 protooncogene has recently emerged. One member of this family, mcl-1, was cloned from a human myeloblastic leukemia cell line (ML-1) undergoing differentiation. The intracellular localization of mcl-1, as well as the kinetics of its expression during differentiation, have now been studied. These studies show that the intracellular distribution of mcl-1 overlaps with, but is not identical to, that of bcl-2:mcl-1 is similar to bcl-2 in that the mcl-1 protein has a prominent mitochondrial localization, and in that it associates with membranes through its carboxyl hydrophobic tail. mcl-1 differs from bcl-2, however, in its relative distribution among other (nonmitochondrial/heavy membrane) compartments, mcl-1 also being abundant in the light membrane fraction of immature ML-1 cells while bcl-2 is abundant in the nuclear fraction. Similarly, in differentiating ML-1 cells, the timing of expression of mcl-1 overlaps with, but is not identical to, that of bcl-2: the mcl-1 protein increases rapidly as cells initiate differentiation, and mcl-1 is a labile protein. In contrast, bcl-2 decreases gradually as cells complete differentiation. Overall, the mcl-1 and bcl-2 proteins have some properties in common and others that are distinct. A burst of expression of mcl-1, prominently associated with mitochondria, complements the continued expression of bcl-2 in ML-1 cells differentiating along the monocyte/macrophage pathway. MCL-1, a member of the bcl-2 family, was cloned from the ML-1 human myeloblastic leukemia cell line (26). ML-1 cells proliferate as immature myeloblasts, and they differentiate to monocytes and macrophages upon exposure to the phorbol ester, 12-0-tetradecanoylphorbol 13-acetate (TPA) I (9). The progression to differentiated phenotype is accompanied by a loss of proliferative capacity without substantial loss of viability, mcl-1 was isolated by screening for genes that increase in expression early in the switch from proliferative to differentiating phenotype (26).It is the carboxyl portion of mcl-1 that has sequence similarity to bcl-2 (the carboxyl 139 out of the 350 amino acid residues of mcl-1 [25]; bcl-2 sequence [8,52]). At the extreme carboxyl terminus, both mcl-1 and bcl-2 have a hydrophobic stretch (19-20 amino acids); in bcl-2, this stretch mediates the association of the protein with membranes (6, 7, 38, 51). bcl-2 was initially reported to localize to mitochondrial membranes and has since also been found in other intraceUular membrane-containing compartments, including the nuclear envelope and endoplasmic reticulum (21,27,33). Recent data show that bcl-2 associates with the outer

To kill invading bacteria, neutrophils must interpret spatial cues, migrate, and reach target sites. Although initiation of chemotactic migration has been extensively studied, little is known about its termination. Here we report that two mitogen-activated protein kinases played opposing roles in neutrophil trafficking. The extracellular signal-regulated kinase (Erk) potentiated G protein-coupled receptor kinase GRK2 activity and inhibited neutrophil migration, whereas p38 MAPK acted as a non-canonical GRK that phosphorylated the formyl peptide receptor FPR1 and facilitated neutrophil migration by blocking GRK2 function. Therefore, the dynamic balance between Erk and p38 MAPK controls neutrophil “stop” and “go” behaviors, ensuring neutrophils precisely reach their final destination as the first line of host-defense.

Hepatitis C virus (HCV) NS5A is a phosphoprotein that possesses a cryptic trans-activation activity. To investigate its potential role in viral replication, we searched for the cellular proteins interacting with NS5A protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a newly discovered soluble N-ethylmaleimide-sensitive factor attachment protein receptor-like protein termed human vesicle-associated membrane protein-associated protein of 33 kDa (hVAP-33). In vitro binding assay and in vivo coimmunoprecipitation studies confirmed the interaction between hVAP-33 and NS5A. Interestingly, hVAP-33 was also shown to interact with NS5B, the viral RNA-dependent RNA polymerase. NS5A and NS5B bind to different domains of hVAP-33: NS5A binds to the C-terminus, whereas NS5B binds to the N-terminus of hVAP-33. Immunofluorescent staining showed a significant colocalization of hVAP-33 with both NS5A and NS5B proteins. hVAP-33 contains a coiled-coil domain followed by a membrane-spanning domain at its C-terminus. Cell fractionation analysis revealed that hVAP-33 is predominantly associated with the ER, the Golgi complex, and the prelysosomal membrane, consistent with its potential role in intracellular membrane trafficking. These interactions provide a mechanism for membrane association of the HCV RNA replication complex and further suggest that NS5A is a part of the viral RNA replication complex.