Physical association between the adipocyte fatty acid-binding protein and hormone-sensitive lipase: a fluorescence resonance energy transfer analysis

Previous in vitro studies have established that hormone sensitive lipase (HSL) and adipocyte fatty acid-binding protein (AFABP) form a physical complex that presumably positions the FABP to accept a product fatty acid generated during catalysis. To assess AFABP-HSL interaction within a cellular context, we have used lipocytes derived from 293 cells (C8PA cells) and examined physical association using fluorescence resonance energy transfer. Transfection of C8PA cells with cyan fluorescent protein (CFP)-HSL, yellow fluorescent protein (YFP)-adipocyte FABP, or YFP-liver FABP revealed that under basal conditions each protein was cytoplasmic. In the presence of 20 microm forskolin, CFP-HSL translocated to the triacylglycerol droplet, coincident with BODIPY-FA labeled depots. Fluorescence resonance energy transfer analysis demonstrated that CFP-HSL associated with YFP-adipocyte FABP in both basal and forskolin-treated cells. In contrast, little if any fluorescence resonance energy transfer could be detected between CFP-HSL and YFP-liver FABP. These results suggest that a pre-lipolysis complex containing at least AFABP and HSL exists and that the complex translocates to the surface of the lipid droplet.     

JNK-dependent release of mitochondrial protein,Smac, during apoptosis in multiple myeloma (MM) cells

Smac, second mitochondria-derived activator of caspases, promotes apoptosis via activation of caspases. Previous studies have shown that c-Jun NH(2)-terminal kinase (JNK) is involved in regulating another mitochondrial protein, cytochrome c during apoptosis; however, the role of JNK in the release of mitochondrial Smac is unknown. Here we show that induction of apoptosis in multiple myeloma (MM) cells is associated with activation of JNK, translocation of JNK from cytosol to mitochondria, and release of Smac from mitochondria to cytosol. Blocking JNK either by dominant-negative mutant (DN-JNK) or cotreatment with a specific JNK inhibitor, SP600125, abrogates both stress-induced release of Smac and induction of apoptosis. These findings demonstrate that activation of JNK is an obligatory event for the release of Smac during stress-induced apoptosis in MM cells.     

Salivary duct carcinoma of the parotid gland: report of a rare case with a comparative study of aspiration cytology and histomorphology

BACKGROUND: Salivary duct carcinomas affecting primarily the parotid gland are extremely rare (0.2-2% of all salivary gland tumors). These carcinomas are considered to be of high grade malignancy, with mortality in up to 70% of cases. They usually affect elderly males and less often young adults. Despite the fact that the histomorphologic characteristics of this tumor are always necessary for its classification, several authors have reported that the cytopathologic approach, using fine needle aspiration (FNA) cytology, can establish the final diagnosis. The aim of this paper is to present a rare case of salivary duct carcinoma of the parotid gland with no typical microscopic findings that was diagnosed by FNA cytology through a combination of techniques on biopsy material. CASE: A 56-year-old male presented with a right parotid mass measuring 6 cm in diameter. The mass appeared to expand subcutaneously and infiltrate the skin of the neck region. Biopsy material from both the mass and skin was obtained using FNA and processed with conventional, cell block and liquid-based cytology techniques. A core biopsy was also performed on the mass for histologic evaluation. CONCLUSION: The findings were consistent with a salivary duct carcinoma of the parotid gland and were confirmed by the histologic report. FNA cytology combined with such techniques as liquid-based cytology provides the potential for the final diagnosis. Liquid-based cytology can improve the cellular morphology of the material and allows immunocytochemistry and other diagnostic techniques. The application of such techniques is significantly restricted by conventional processing; thus, combining liquid-based cytology with other techniques expands the boundaries of cytology as a diagnostic test.     

Post-translational regulation of adipose differentiation-related protein by the ubiquitin/proteasome pathway

Adipose differentiation-related protein (ADRP) is localized to lipid droplets in most mammalian cells. ADRP, proposed to regulate fatty acid mobilization and lipid droplet formation, is linked to lipid accumulation in foam cells of human atherosclerotic lesions. In this report, we show that ADRP protein accumulates in Chinese hamster ovary fibroblastic cells cultured in the presence of oleic acid but is destabilized when fatty acid sources are removed from culture serum. The latter effect was blocked by the proteasome inhibitor MG132, whereas inhibitors of other proteolytic processes were ineffective. Pulse-chase experiments confirmed that ADRP degradation is inhibited by MG132. Conditions that stimulate ADRP degradation also promoted the covalent modification of ADRP by ubiquitin, whereas the addition of oleic acid to culture media, which promotes triacylglycerol deposition, blunted the appearance of ubiquitinated-ADRP. Treatment with MG132 increased the levels of ADRP associated with lipid droplets, as well as throughout the cytosol. Finally, we demonstrate that the disappearance of ADRP protein after the onset of perilipin expression during adipocyte differentiation is due to degradation by proteasomes Thus, proteolytic degradation of ADRP mediated through the ubiquitin/proteasome pathway appears to be a major mode for the post-translational regulation of ADRP.     

Non-transfer of materials between plasmodia of different species of myxomycetes

Summary Under the experimental conditions of this study no transfer of radiophosphorus occurred from one living plasmodium to another when a radioactive plasmodium ofPhysarum polycephalum and a non-radioactive plasmodium ofPhysarum gyrosum orFuligo septica were in intimate contact for 24 hours.This research constitutes a part of a program Experimental Approach to the Taxonomy of the Myxomycetes, supported by National Science Foundation Grant G-6382.     

Molecular interaction between organophosphorus acid anhydrolase and diisopropylfluorophosphate

Organophosphorus acid anhydrolases (OPAA; E.C.3.1.8.2) are a class of enzymes that hydrolyze a variety of toxic acetylcholinesterase-inhibiting organophosphorus (OP) compounds, including pesticides and fluorine-containing chemical nerve agents. In this paper, subphase conditions have been optimized to obtain stable OPAA Langmuir films, and the diisopropylfluorophosphate (DFP) hydrolysis reaction catalyzed by OPAA in aqueous solution and at the air-water interface was studied. OPAA-DFP interactions were investigated utilizing different spectroscopic techniques, that is, circular dichroism and fluorescence in aqueous solution and infrared reflection absorption spectroscopies at the air-water interface. The characterization of OPAA and its secondary structure in aqueous solution and as a monolayer at the air-water interface in the absence and in the presence of DFP dissolved in aqueous solution or in the aqueous subphase demonstrated significantly distinctive features. The research described herein demonstrated that OPAA can be used in an enzyme-based biosensor for DFP detection.     

RIAM, an Ena/VASP and Profilin ligand, interacts with Rap1-GTP and mediates Rap1-induced adhesion

The small GTPase Rap1 induces integrin-mediated adhesion and changes in the actin cytoskeleton. The mechanisms that mediate these effects of Rap1 are poorly understood. We have identified RIAM as a Rap1-GTP-interacting adaptor molecule. RIAM defines a family of adaptor molecules that contain a RA-like (Ras association) domain, a PH (pleckstrin homology) domain, and various proline-rich motifs. RIAM also interacts with Profilin and Ena/VASP proteins, molecules that regulate actin dynamics. Overexpression of RIAM induced cell spreading and lamellipodia formation, changes that require actin polymerization. In contrast, RIAM knockdown cells had reduced content of polymerized actin. RIAM overexpression also induced integrin activation and cell adhesion. RIAM knockdown displaced Rap1-GTP from the plasma membrane and abrogated Rap1-induced adhesion. Thus, RIAM links Rap1 to integrin activation and plays a role in regulating actin dynamics.