Agonist-dependent traffic of raft-associated Ras and Raf-1 is required for activation of the mitogen-activated protein kinase cascade

Stimulation of HIRcB fibroblasts with insulin leads to accumulation of active components of the mitogen-activated protein kinase cascade in endocytic compartments. However, the factors that regulate the mobilization of these components through the endocytic pathway and the relevance of this event to cellular signaling remain unclear. Here we report that Ras proteins are associated with lipid rafts in resting HIRcB fibroblasts. Ras is rapidly internalized into the endocytic compartment following stimulation with insulin. The redistribution of Ras is independent of its activation. Attachment of the C-terminal 20 amino acids of Ha-Ras to green fluorescent protein was sufficient to target this construct to the same loci as the endogenous Ras protein, indicating that Ras distribution is a consequence of the association of its lipid modified C terminus with membranes. Depletion of plasma membrane cholesterol delocalized Ras and blocked insulin-dependent Ras traffic. Cholesterol depletion also blocked insulin-dependent phosphorylation of MEK and mitogen-activated protein kinase (MAPK) but had no effects on the translocation and activation of Raf-1. A second inhibitor of endocytosis, cytochalasin D, also blocked insulin-dependent MAPK phosphorylation. Taken together, these results suggest that mobilization of active Raf-1 through the endocytic compartment is required for completion of the MAPK cascade.     

Adenovirus-mediated expression of Fas ligand induces apoptosis of human prostate cancer cells

Several laboratories have reported on the apoptotic potentials of human prostate cancer (PC) cell lines in response to crosslinking of Fas (CD95/APO-1) with agonistic anti-Fas antibodies. We have re-evaluated the apoptotic potentials of seven human PC cell lines using the natural Fas ligand (FasL) in place of agonistic antibody. First, PC cell lines were tested in a standard cytotoxicity assay with a transfected cell line that stably expresses human FasL. Next, we developed an adenoviral expression system employing 293 cells that stably express crmA, a poxvirus inhibitor of apoptosis, to analyze the effects of FasL when expressed internally by the PC cell lines. Our data suggest that the apoptotic potentials of these cell lines were greatly underestimated in previous studies utilizing agonistic anti-Fas antibodies. Lastly, adenoviral-mediated expression of FasL prevented growth and induced regression of two human PC cell lines in immunodeficient mice. These preliminary in vivo results suggest a potential use for adenovirus encoding FasL as a gene therapy for PC.     

Modified approach for apoptosis detection reveals changes in apoptotic processes in the seminoma-associated tissue

Apoptosis morphology (DNA condensation) and internucleosomal DNA cleavage (TdT assay) were measured simultaneously on double fluorescence labeled testis tumor sections, employing conventional immunofluores cence microscopy. Six different apoptosis indices (AI) were determined based either solely on morphological or biochemical criteria, or on a combination of both processes. Measurements were performed in metastasized and non-metastasized seminoma, and in histological regions located distantly and associated with the tumor. Preliminary results on 19 histologies revealed that up to 66% of apoptotic cells were not detected, depending on the method used for apoptosis detection. Besides, no changes of solely morphologically defined AI was found in the different histological regions. By contrast, significant changes (p < 0.0004) in the different histological regions were detected when measuring AIs, e.g., defined by DNA fragmentation occuring without DNA condensation in apoptotic cells. Those changes were not detected in metastasized seminoma. These data, for the first time allow a comparison of two widely used approaches for apoptosis detection. Furthermore, the results reveal differences in apoptotic processes in tissue associated with non-metastasized seminoma detectable by a modified evaluated TdT assay but not by morphological changes, although this TdT method fails to show the total amount of apoptotic cells.     

The effect of thin filament activation on the attachment of weak binding cross-bridges: A two-dimensional x-ray diffraction study on single muscle fibers

To study possible structural changes in weak cross-bridge attachment to actin upon activation of the thin filament, two-dimensional (2D) x-ray diffraction patterns of skinned fibers from rabbit psoas muscle were recorded at low and high calcium concentration in the presence of saturating concentrations of MgATPgammaS, a nucleotide analog for weak binding states. We also studied 2D x-ray diffraction patterns recorded under relaxing conditions at an ionic strength above and below 50 mM, because it had been proposed from solution studies that reducing ionic strength below 50 mM also induces activation of the thin filament. For this project a novel preparation had to be established that allows recording of 2D x-ray diffraction patterns from single muscle fibers instead of natural fiber bundles. This was required to minimize substrate depletion or product accumulation within the fibers. When the calcium concentration was raised, the diffraction patterns recorded with MgATPgammaS revealed small changes in meridional reflections and layer line intensities that could be attributed in part to the effects of calcium binding to the thin filament (increase in I380, decrease in first actin layer line intensity, increase in I59) and in part to small structural changes of weakly attached cross-bridges (e.g., increase in I143 and I72). Calcium-induced small-scale structural rearrangements of cross-bridges weakly attached to actin in the presence of MgATPgammaS are consistent with our previous observation of reduced rate constants for attachment and detachment of cross-bridges with MgATPgammaS at high calcium. Yet, no evidence was found that weakly attached cross-bridges change their mode of attachment toward a stereospecific conformation when the actin filament is activated by adding calcium. Similarly, reducing ionic strength to less than 50 mM does not induce a transition from nonstereospecific to stereospecific attachment.     

Human DU145 prostate cancer cells overexpressing mitogen-activated protein kinase phosphatase-1 are resistant to Fas ligand-induced mitochondrial perturbations and cellular apoptosis

Recent studies have suggested that MAP kinase phosphatase 1 (MKP-1) is overexpressed in prostate cancer. To evaluate the role of MKP-1 in regulating cell death and tumor growth in prostate cancer, MKP-1 was conditionally overexpressed in the human prostate cancer cell line DU145. Overexpression of MKP-1 in DU145 cells blocked activation of stress-activated protein kinase (SAPK/JNK). MKP-1 overexpression in DU-145 cells was also found to inhibit Fas ligand (FasL)-induced apoptosis, as well as block the activation of caspases by Fas engagement. In addition, MKP-1 blocked the activation of apoptosis by transfected MEKK-1 and ASK-1, presumably through its inhibition of the SAPK/JNK family of enzymes. MKP-1 blocked the ability of FasL to induce loss of mitochondrial transmembrane potential (_m), suggesting that MKP-1 acts upstream of mitochondrial pro-apoptotic events induced by FasL and that the SAPK/JNK pathway may form the signaling link between Fas receptor and mitochondrial dysfunction. Thus, MKP-1 overexpression in prostate cancer may play a role in promoting prostate carcinogenesis by inhibiting FasL-induced cell death.     

'Brain aided' musk design

This brief review, including new experimental results, is the summary of a talk at the RSC/SCI conference flavours & fragrances 2004 in Manchester, United Kingdom, 12-14 May, 2004. Musk odorants have been a classical domain for computer aided structure-odor relationship (SOR) studies, but, contrary to sandalwood or amber odorants, they belong to structurally very different substance classes, e.g., macrocycles, aromatic polycycles, and nitro arenes. Most SOR computer models are restricted to one class, excluding structural diversity to increase predictability. But even within a musk family, structural similarities are often due to a common synthetic access, and do not reflect binding requirements for the musk receptor. Beyond that, the importance of structural key features can be missed, which is discussed on the example of the (4S)-Me group of Galaxolide. By synthesis and olfactory evaluation of Galaxolide-like shaped macrobicycles as model compounds for conformationally constrained (12R)-12-methyltridecano-13-lactone, it was investigated how likely there is more than one musk receptor. Finally, the new family of so-called linear musks is discussed, especially with respect to the conformational importance of the gem-2',2'-dimethyl moiety in Helvetolide and the additional 2'-carbonyl group of Romandolide--structural features that strongly diminish the musk odor of macrocycles. On the example of 2-methyl-2-[(E)-1,2,4-trimethylpent-2-enyloxy]propyl esters, the 'brain-aided' design and conformational analysis of musk odorants is illustrated. The overview concludes with the synthesis, odor evaluation, and conformational discussion of the new musk odorant 2-(3,3-dimethylcyclohexyl)propanoic acid ethoxycarbonylmethyl ester.     

The anaphase promoting complex/cyclosome is recruited to centromeres by the spindle assembly checkpoint

The anaphase promoting complex/cyclosome (APC/C) is crucial to the control of cell division (for a review, see ref. 1). It is a multi-subunit ubiquitin ligase that, at defined points during mitosis, targets specific proteins for proteasomal degradation. The APC/C is itself regulated by the spindle or kinetochore checkpoint, which has an important role in maintaining genomic stability by preventing sister chromatid separation until all chromosomes are correctly aligned on the mitotic spindle. The spindle checkpoint regulates the APC/C by inactivating Cdc20, an important co-activator of the APC/C. There is also evidence to indicate that the spindle checkpoint components and Cdc20 are spatially regulated by the mitotic apparatus, in particular they are recruited to improperly attached kinetochores. Here, we show that the APC/C itself co-localizes with components of the spindle checkpoint to improperly attached kinetochores. Indeed, we provide evidence that the spindle checkpoint machinery is required to recruit the APC/C to kinetochores. Our data indicate that the APC/C could be regulated directly by the spindle checkpoint.     

Plasma distribution of apoA-IV in patients with coronary artery disease and healthy controls

Recent studies showed lower apolipoprotein A-IV (apoA-IV) plasma concentrations in patients with coronary artery disease (CAD). The actual distribution of the antiatherogenic apoA-IV in human plasma, however, is discussed controversially and it was never investigated in CAD patients. We therefore developed a gentle technique to separate the various apoA-IV-containing plasma fractions. Using a combination of precipitation of all lipoproteins with 40% phosphotungstic acid and 4 M MgCl2, as well as immunoprecipitation of all apoA-I-containing particles with an anti-apoA-I antibody, we obtained three fractions of apoA-IV: lipid-free apoA-IV (about 4% of total apoA-IV), apoA-IV associated with apoA-I (LpA-I:A-IV, 12%), and apoA-I-unbound but lipoprotein-containing apoA-IV (LpA-IV, 84%). We compared these three apoA-IV fractions between 52 patients with a history of CAD and 52 age- and sex-matched healthy controls. Patients had significantly lower apoA-IV levels when compared to controls (10.28 +/- 3.67 mg/dl vs. 11.85 +/- 2.82 mg/dl, P = 0.029), but no major differences for the three plasma apoA-IV fractions. We conclude that our gentle separation method reveals a different distribution of apoA-IV than in many earlier studies. No major differences exist in the apoA-IV plasma distribution pattern between CAD patients and controls. Therefore, the antiatherogenic effect of apoA-IV has to be explained by other functional properties of apoA-IV (e.g., the antioxidative characteristics).     

Sulfur oxidation in Paracoccus pantotrophus: interaction of the sulfur-binding protein SoxYZ with the dimanganese SoxB protein

The central protein of the sulfur-oxidizing enzyme system of Paracoccus pantotrophus, SoxYZ, formed complexes with subunits associated and covalently bound. In denaturing SDS-polyacrylamide gel electrophoresis (PAGE) SoxY migrated at 12 and SoxZ at 16kDa. SDS-PAGE of homogeneous SoxYZ without reductant separated dimeric complexes of 25, 29, and 32kDa identified by the N-terminal amino acid sequences as SoxY-Y, SoxY-Z, and SoxZ-Z, and subunit cleavage by reduction suggested their linkage via protein disulfide bonds. SoxYZ was reversibly redox active between -0.25 and 0.2V, as monitored by a combined electrochemical and FTIR spectroscopic approach. The dimanganese SoxB protein (58.611Da) converted the covalently linked heterodimer SoxY-Z to SoxYZ with associated subunits which in turn aggregated to the heterotetramer Sox(YZ)(2). This reaction depended on time and the SoxB concentration, and demonstrated the interaction of these two Sox proteins.     

Exploring complex fitness surfaces: multiple ornamentation and polymorphism in male guppies

Abstract.— The sexual ornamentation used by male guppies to attract females comprises many components, each of which varies considerably among males. Although natural and sexual selection have been shown to contribute to divergence among populations in male sexual ornaments, the role of sexual selection in maintaining polymorphism within populations is less clear. We used both parametric quadratic regression and nonparametric projection pursuit regression techniques to reveal the major axes of non-linear sexual selection on male ornaments. We visualized the fitness surfaces defined by these axes using thin-plate splines to allow a direct comparison of the two methodologies. Identification of the major axes of selection and their visualization was critical in determining the form and strength of nonlinear selection. Both types of analysis revealed fitness surfaces comprising three peaks, suggesting that there is more than one way to make an attractive guppy. Disruptive selection may be an important process underlying the presence of multiple sexual ornaments and may contribute to the maintenance of the high levels of polymorphism in male sexual ornaments found in guppy populations.