DHA induces ER stress and growth arrest in human colon cancer cells: associations with cholesterol and calcium homeostasis

Polyunsaturated fatty acids (PUFAs) are normal constituents of the diet, but have properties different from other fatty acids (e.g., through generation of signaling molecules). N-3 PUFAs reduce cancer cell growth, but no unified mechanism has been identified. We show that docosahexaenoic acid (DHA; 22:6 n-3) causes extensive changes in gene expression patterns at mRNA level in the colon cancer cell line SW620. Early changes include unfolded protein response (UPR) and increased levels of phosphorylated eIF2alpha as verified at protein level. The latter is considered a hallmark of endoplasmic reticulum (ER) stress and is abundantly present already after 3 h. It may coordinate many of the downstream changes observed, including signaling pathways for cell cycle arrest/apoptosis, calcium homeostasis, cholesterol metabolism, ubiquitination, and proteasomal degradation. Also, eicosapentaenoic acid (EPA), but not oleic acid (OA), induced key mediators of ER stress and UPR at protein level. Accumulation of esterified cholesterol was not compensated for by increased total levels of cholesterol, and mRNAs for cholesterol biosynthesis as well as de novo synthesis of cholesterol were reduced. These results suggest that cytotoxic effects of DHA are associated with signaling pathways involving lipid metabolism and ER stress.     

Explosive increase in ectoparasites in Hawaiian forest birds

Ectoparasites, particularly chewing lice in the Phthiraptera (Insecta), affect the ecology of numerous host species. Most lice are highly host-specific, and there are no documented cases of major increases of chewing lice, within populations, over years. During continuous study from 1987-2005 at upper elevation forests on the island of Hawaii, chewing lice were exceedingly rare and, until 2003, were found in just 2 of 12 species of native and introduced birds. From 2003-2005, there was an explosive increase in the prevalence of chewing lice in all host species. There was no change in humidity, or in behavior of hosts, that could have caused an ecological release of existing lice. Based on reduced fat levels and increases in broken wing and tail feathers for most host species, there was apparently a food limitation that preceded the increase. The increase coincided temporally with detection of a nonnative bird that had recently been found in elevations below the study sites. Although there were isolated sightings of this bird on the study sites, seasonal movements and behavior of some species of native birds could also have allowed greater transmission to study sites. Both prevalence and intensity of infection, indexed by number of body regions parasitized, were lower in native species with greater bill overlap, a character that could help birds control lice. Seasonality of prevalence indicated that low prevalence preceded molt and high prevalence occurred after molting of hosts. The number of major fault bars in wing and tail feathers, a sign of nutritive stress, was correlated with intensity of infection, indicating an indirect cost to the hosts of being parasitized. In addition, birds with lice were less likely to be recaptured than birds without lice.     

Enhanced detection and identification of multiply phosphorylated peptides using TiO2 enrichment in combination with MALDI TOF/TOF MS

The analysis of PTMs such as phosphorylation has become an important field in MS because they can directly indicate protein states and interactions. Whereas the characterization of singly and doubly phosphorylated peptides has almost become routine, identifying phosphorylation events at multiple residues within a small region of a protein is still problematic. The identification of multiple modifications can be further hampered by low sequence information due to multiple neutral losses from phosphorylated side chains. Here we present a strategy for the analysis of complex phosphopeptides that combines peptide enrichment by titanium dioxide, separation by RP separation on monolithic columns and MS using high energy HE-CAD in a MALDI TOF/TOF analyser. Using synthetic phosphopeptides our approach is compared to multistage activation (MSA) MS/MS and the recently described electron transfer dissociation (ETD) method using an ESI-LTQ mass spectrometer.     

Extrakorporale Membranoxygenierung in der Intensivmedizin

Extracorporeal membrane oxygenation (ECMO) represents a valuable tool in the armamentarium of life and organ support measures in intensive care medicine. ECMO is used in fulminant pulmonary failure to prevent acute hypoxia and to allow CO₂ removal (veno-venous ECMO) or, in highly selected cases of acute cardiovascular failure, to maintain organ perfusion and gas exchange and to prevent imminent death (veno-arterial ECMO). The purpose of ECMO is to allow time for intrinsic recovery of the lungs and heart. Alternatively, ECMO can be used as bridging device until a suitable organ for transplantation is available or the implantation of an artificial heart is feasible. ECMO carries a high complication rate and its use should be limited to highly specialized centers. In rare cases, mobile ECMO systems can be used by dedicated teams which allow transportation of critically ill patients over longer distances to specialized institutions. There is no unequivocal evidence from controlled studies that the use of ECMO in adults improves survival in larger cohorts, but its use in selected cases can be life saving. Long-term survivors of ECMO therapy report significant impairments in health-related quality of life (HRQL) when compared to healthy controls. There is, however, a gradual recovery and improvement in HRQL over a prolonged period after discharge from the intensive care unit and severe limitations in cognitive or physical function are rare. The incidence of chronic post-traumatic stress disorder (PTSD) in patients after ECMO can reach up to 30%. PTSD has a major negative impact on HRQL outcomes of ECMO therapy. PTSD or other stress-related disorders need to be diagnosed and adequately treated to allow adequate recovery from the extreme illness these patients have survived.     

Polyisoprenoid epoxides stimulate the biosynthesis of coenzyme Q and inhibit cholesterol synthesis

In our search for compounds that up-regulate the biosynthesis of coenzyme Q (CoQ), we discovered that irradiation of CoQ with ultraviolet light results in the formation of a number of compounds that influence the synthesis of mevalonate pathway lipids by HepG2 cells. Among the compounds that potently stimulated CoQ synthesis while inhibiting cholesterol synthesis, derivatives of CoQ containing 1-4 epoxide moieties in their polyisoprenoid side chains were identified. Subsequently, chemical epoxidation of all-trans-polyprenols of different lengths revealed that the shorter farnesol and geranylgeraniol derivatives were without effect, whereas the longer derivatives of solanesol enhanced CoQ and markedly reduced cholesterol biosynthesis. In contrast, none of the modified trans-trans-poly-cis-polyprenols exerted noticeable effects. Tocotrienol epoxides were especially potent in our system; those with one epoxide moiety in the side-chain generally up-regulated CoQ biosynthesis by 200-300%, whereas those with two such moieties also decreased cholesterol synthesis by 50-90%. Prolonged treatment of HepG2 cells with tocotrienol epoxides for 26 days elevated their content of CoQ by 30%. In addition, the levels of mRNA encoding enzymes involved in CoQ biosynthesis were also elevated by the tocotrienol epoxides. The site of inhibition of cholesterol synthesis was shown to be oxidosqualene cyclase. In conclusion, epoxide derivatives of certain all-trans-polyisoprenoids cause pronounced stimulation of CoQ synthesis and, in some cases, simultaneous reduction of cholesterol biosynthesis by HepG2 cells.     

Investigation of coenzyme Q biosynthesis in human fibroblast and HepG2 cells

Coenzyme Q (CoQ) deficiency occurs in genetic disorders, during aging and various diseases. Diagnosis requires skin fibroblasts in tissue culture. [³H]Mevalonate incorporation was appropriate to measure the rate of CoQ synthesis in fibroblasts and hepatoblastoma cells. [¹⁴C]p-Hydroxybenzoate had limited permeability, but it could be increased with Fugene and cyclodextrin. Inhibition of decaprenyl-4-hydroxybenzoate transferase results in the accumulation of decaprenyl diphosphate, an indicator of enzyme deficiency. Also, analysis of the corresponding mRNAs in this case is useful. In vitro assays to measure trans-prenyltransferase and decaprenyl-4-hydroxybenzoate transferase activities are not available. Neither measurement of methyltransferases is reliable in human cells. In vitro reconstruction of CoQ synthesis, in opposite to cholesterol synthesis, proved to be unsuccessful. Thus, the biochemical characterization of the CoQ biosynthetic system in human cells is restricted to a few reliable analytical procedures.     

Towards new enzymes for biofuels: lessons from chitinase research

Enzymatic conversion of structural polysaccharides in plant biomass is a key issue in the development of second generation ('lignocellulosic') bioethanol. The efficiency of this process depends in part on the ability of enzymes to disrupt crystalline polysaccharides, thus gaining access to single polymer chains. Recently, new insights into how enzymes accomplish this have been obtained from studies on enzymatic conversion of chitin. First, chitinolytic microorganisms were shown to produce non-hydrolytic accessory proteins that increase enzyme efficiency. Second, it was shown that a processive mechanism, which is generally considered favorable because it improves substrate accessibility, might in fact slow down enzymes. These findings suggest new focal points for the development of enzyme technology for depolymerizing recalcitrant polysaccharide biomass. Improving substrate accessibility should be a key issue because this might reduce the need for using processive enzymes, which are intrinsically slow and abundantly present in current commercial enzyme preparations for biomass conversion. Furthermore, carefully selected substrate-disrupting accessory proteins or domains might provide novel tools to improve substrate accessibility and thus contribute to more efficient enzymatic processes.     

Expression and characterization of endochitinase C from Serratia marcescens BJL200 and its purification by a one-step general chitinase purification method

In this study we cloned, expressed, purified, and charaterized chitinase C1 from Serratia marcescens strain BJL200. As expected, the BJL200-ChiC1 amino acid sequence of this strain was highly similar to sequences of ChiC1 identified in two other strains of S. marcescens. BJL200-ChiC1 was overproduced in E. coli by the T7 expression system, and purified by a one-step hydrophobic interaction chromatography (HIC) with phenyl-sepharose. BJL200-ChiA and BJL200-ChiB had an approximately 30-fold higher k(cat) and 15 fold-lower K(m) than BJL200-ChiC1 for the oligomeric substrate 4-methylumbelliferyl-beta-D-N-N'-N'-triacetylchitotrioside, while BJL200-ChiC1 was 10-15 times faster than BJL200-ChiB and BJL200-ChiA in degrading the polymeric substrate CM-chitin-RBV. BJL200-ChiC1 degradation of beta-chitin resulted in a range of different chito-oligosaccharides (GlcNAc)(2) (main product), GlcNAc, (GlcNAc)(3), (GlcNAc)(4), and (GlcNAc)(5), indicating endo activity. The purification method used for BJL200-ChiC1 in this study is generally applicable to family 18 chitinases and their mutants, including inactive mutants, some of which tend to bind almost irreversibly to chitin columns. The high specificity of the interaction with the (non-chitinous) column material is mediated by aromatic residues that occur in the substrate-binding clefts and surfaces of the enzymes.