The NF2 tumor suppressor gene product, merlin, inhibits cell proliferation and cell cycle progression by repressing cyclin D1 expression

Inactivation of the NF2 tumor suppressor gene has been observed in certain benign and malignant tumors. Recent studies have demonstrated that merlin, the product of the NF2 gene, is regulated by Rac/PAK signaling. However, the mechanism by which merlin acts as a tumor suppressor has remained obscure. In this report, we show that adenovirus-mediated expression of merlin in NF2-deficient tumor cells inhibits cell proliferation and arrests cells at G1 phase, concomitant with decreased expression of cyclin D1, inhibition of CDK4 activity, and dephosphorylation of pRB. The effect of merlin on cell cycle progression was partially overridden by ectopic expression of cyclin D1. RNA interference experiments showed that silencing of the endogenous NF2 gene results in upregulation of cyclin D1 and S-phase entry. Furthermore, PAK1-stimulated cyclin D1 promoter activity was repressed by cotransfection of NF2, and PAK activity was inhibited by expression of merlin. Interestingly, the S518A mutant form of merlin, which is refractory to phosphorylation by PAK, was more efficient than the wild-type protein in inhibiting cell cycle progression and in repressing cyclin D1 promoter activity. Collectively, our data indicate that merlin exerts its antiproliferative effect, at least in part, via repression of PAK-induced cyclin D1 expression, suggesting a unifying mechanism by which merlin inactivation might contribute to the overgrowth seen in both noninvasive and malignant tumors.     

Syntheses, crystal structure and cytotoxicity of diamine platinum(II) complexes containing maltol

The cationic complexes (1,2-diaminoethane)(maltolato)platinum(II) ([Pt(en)(ma)]+) and (1R,2R-1,2-diaminocyclohexane)(maltolato)platinum(II) ([Pt(R,R-DACH)(ma)]+) have been prepared and the structure of [Pt(R,R-DACH)(ma)]NO3 has been determined by single crystal X-ray diffraction. The geometry of the metal in [Pt(R,R-DACH)(ma)]NO3 is essentially square planar and the maltolate ligand has a geometry similar to other chelate complexes involving this ligand. The cytotoxicities of the compounds have been assessed in the human cell lines HeLa and K562 and the IC50 values are approximately 32 microM in HeLa cells and 26 microM in K562 cells. In these cell lines the cytotoxicity of cisplatin is higher than the maltolate complexes by a factor of 2 to 3 whereas the cytotoxicity of carboplatin is lower than the maltolate complexes.     

Physical fitness of an industrial fire department vs. a municipal fire department

Both industrial and municipal firefighters need to maintain high levels of physical fitness and minimize cardiovascular risk factors. The nature of firefighter responsibilities in industrial and municipal settings may vary, affecting the ability to sustain high levels of physical fitness. We compared the working conditions, physical fitness, and exercise training practices of an industrial fire department (n = 17) to those of a nearby municipal fire department (n = 55). After informed consent, aerobic capacity, muscular strength, muscular endurance, body composition, flexibility, blood lipid concentrations, and blood pressure levels were measured. Exercise training practices and related factors were assessed using a questionnaire. Despite programmatic differences, these departments demonstrated similar, relatively high degrees of physical fitness and similar blood lipid concentrations, blood pressure levels, and cardiac risk factors. It is recommended that fire departments involve appropriately trained staff, schedule on-duty times for exercise, offer well-equipped exercise facilities, and follow National Strength and Conditioning Association (NSCA) and American College of Sports Medicine (ACSM) guidelines for exercise conditioning in order to maintain a high degree of physical fitness.     

Synthesis and cytotoxicity of salicylate-based poly(anhydride esters)

This paper describes the synthesis and cytotoxicity of poly(anhydride esters) that are composed of several salicylate derivatives, including halogenated salicylates, aminosalicylates, salicylsalicylic acid, and thiolsalicylic acid. The incorporation of these nonsteroidal antiinflammatory drugs (NSAIDs) into a biodegradable polymer backbone yields drug-based polymers that have potential for a variety of applications. The poly(anhydride esters) were synthesized by melt condensation polymerization. The halogenated salicylate derivatives yielded the highest molecular polymers as well as the highest glass transition temperatures. All polymers displayed in vitro degradation lag times from 1 to 3 days, depending on the water solubility of the salicylate derivative. Cell viability and proliferation were determined with L929 fibroblast cells in serum-containing medium to assess the polymer cytotoxicities, which varied as a function of the saliyclate chemistry. Cell morphology was normal for most of the polymers evaluated.     

Chemical synthesis of (S)-4,5-dihydroxy-2,3-pentanedione, a bacterial signal molecule precursor, and validation of its activity in Salmonella typhimurium

We describe an original, short, and convenient chemical synthesis of enantiopure (S)-4,5-dihydroxy-2,3-pentanedione (DPD), starting from commercial methyl (S)-(-)-2,2-dimethyl-1,3-dioxolane-4-carboxylate. DPD is the precursor of autoinducer (AI)-2, the proposed signal for bacterial interspecies communication. AI-2 is synthesized by many bacterial species in three enzymatic steps. The last step, a LuxS-catalyzed reaction, leads to the formation of DPD, which spontaneously cyclizes into AI-2. AI-2-like activity of the synthesized molecule was ascertained by the Vibrio harveyi bioassay. To further validate the biological activity of synthetic DPD and to explore its potential in studying DPD (AI-2)-mediated signaling, a Salmonella typhimurium luxS mutant was constructed. Expression of the AI-2 regulated lsr operon can be rescued in this luxS mutant by addition of synthetic DPD or genetic complementation. Biofilm formation by S. typhimurium has been reported to be defective in a luxS mutant, and this was confirmed in this study to test DPD for chemical complementation. However, biofilm formation of the luxS mutant cannot be restored by addition of DPD. In contrast, introduction of luxS under control of its own promoter complemented biofilm formation. Further results demonstrated that biofilm formation of the luxS mutant cannot be restored with luxS under control of the strong nptII promoter. This indicates that altering the intrinsic promoter activity of luxS affects Salmonella biofilm formation. Conclusively, we synthesized biologically active DPD. Using this chemical compound in combination with genetic approaches opens new avenues in studying AI-2-mediated signaling.     

Omega-oxidation of 20-hydroxyeicosatetraenoic acid (20-HETE) in cerebral microvascular smooth muscle and endothelium by alcohol dehydrogenase 4

20-Carboxyeicosatetraenoic acid (20-COOH-AA) is a bioactive metabolite of 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid that produces vasoconstriction in the cerebral circulation. We found that smooth muscle (MSMC) and endothelial (MEC) cultures obtained from mouse brain microvessels convert [3H]20-HETE to 20-COOH-AA, indicating that the cerebral vasculature can produce this metabolite. The [3H]20-COOH-AA accumulated primarily in the culture medium, together with additional radiolabeled metabolites identified as the chain-shortened dicarboxylic acids 18-COOH-18:4, 18-COOH-18:3, and 16-COOH-16:3. N-Heptylformamide, a potent inhibitor of alcohol dehydrogenase (ADH), decreased the conversion of [3H]20-HETE to 20-COOH-AA by the MSMC and MEC and also by isolated mouse brain microvessels. Purified mouse and human ADH4, human ADH3, and horse liver ADH1 efficiently oxidized 20-HETE, and ADH4 and ADH3 were detected in MSMC and MEC by Western blotting. N-Heptylformamide inhibited the oxidation of 20-HETE by mouse and human ADH4 but not by ADH3. These results demonstrated that cerebral microvessels convert 20-HETE to 20-COOH-AA and that ADH catalyzes the reaction. Although ADH4 and ADH3 are expressed in MSMC and MEC, the inhibition produced by N-heptylformamide suggests that ADH4 is primarily responsible for 20-COOH-AA formation in the cerebral microvasculature.     

Quantitative measurements of Ca(2+)/calmodulin binding and activation of myosin light chain kinase in cells

Myosin II regulatory light chain (RLC) phosphorylation by Ca(2+)/calmodulin (CaM)-dependent myosin light chain kinase (MLCK) is implicated in many cellular actin cytoskeletal functions. We examined MLCK activation quantitatively with a fluorescent biosensor MLCK where Ca(2+)-dependent increases in kinase activity were coincident with decreases in fluorescence resonance energy transfer (FRET) in vitro. In cells stably transfected with CaM sensor MLCK, increasing [Ca(2+)](i) increased MLCK activation and RLC phosphorylation coincidently. There was no evidence for CaM binding but not activating MLCK at low [Ca(2+)](i). At saturating [Ca(2+)](i) MLCK was not fully activated probably due to limited availability of cellular Ca(2+)/CaM.     

An Arabidopsis RNA lariat debranching enzyme is essential for embryogenesis

An embryo-defective mutant of Arabidopsis thaliana was isolated that arrests development at a variety of stages, from as early as the globular stage of embryogenesis to as late as formation of an abnormal bent cotyledon stage embryo. Defects in the suspensor, a normally transient structure derived from the fertilized egg, were often associated with the arrested embryo. The lesion was within a gene encoding a protein with domains characteristic of lariat debranching enzymes, which has been named AtDBR1 (for Arabidopsis thaliana Debranching enzyme 1). Cleavage of the 2'-5'-phosphodiester bond found in excised intron lariats ("debranching") is essential for turnover of intronic sequences as well as generation of some small nucleolar RNAs. The mutation within AtDBR1 was confirmed by complementation as being responsible for the embryo-lethal phenotype, and the activity of the encoded protein in cleavage of 2'-5'-phosphodiester bonds was verified using an in vitro debranching assay.