Disposable real-time microPCR device: lab-on-a-chip at a low cost

We have designed, fabricated and tested a real-time micro polymerase chain reaction (microPCR) system. It consists of a microscope glass cover slip placed on top of a micromachined silicon chip integrated with a heater and a temperature sensor. A single microL of a sample containing DNA was placed on the glass and encapsulated with mineral oil to prevent the evaporation of water, thus forming a virtual reaction chamber (VRC). The PCR chip required half a second to heat up from 72 to 94 degrees C and two seconds to cool from 94 to 55 degrees C, corresponding to a cooling rate of -20 K s(-1). The real-time PCR yield was determined by a fluorescence method. The melting curve analysis method as well as capillary electrophoresis was performed to determine the purity of the PCR product. As the glass slip is disposable, cross-contamination from sample to sample is eliminated. The total cost of running the PCR is given by the value of the cover slip and its treatment.     

A common lipid links Mfn-mediated mitochondrial fusion and SNARE-regulated exocytosis

Fusion of vesicles into target membranes during many types of regulated exocytosis requires both SNARE-complex proteins and fusogenic lipids, such as phosphatidic acid. Mitochondrial fusion is less well understood but distinct, as it is mediated instead by the protein Mitofusin (Mfn). Here, we identify an ancestral member of the phospholipase D (PLD) superfamily of lipid-modifying enzymes that is required for mitochondrial fusion. Mitochondrial PLD (MitoPLD) targets to the external face of mitochondria and promotes trans-mitochondrial membrane adherence in a Mfn-dependent manner by hydrolysing cardiolipin to generate phosphatidic acid. These findings reveal that although mitochondrial fusion and regulated exocytic fusion are mediated by distinct sets of protein machinery, the underlying processes are unexpectedly linked by the generation of a common fusogenic lipid. Moreover, our findings suggest a novel basis for the mitochondrial fragmentation observed during apoptosis.     

Filopodia formation in the absence of functional WAVE- and Arp2/3-complexes

Cell migration is initiated by plasma membrane protrusions, in the form of lamellipodia and filopodia. The latter rod-like projections may exert sensory functions and are found in organisms as distant in evolution as mammals and amoeba such as Dictyostelium discoideum. In mammals, lamellipodia protrusion downstream of the small GTPase Rac1 requires a multimeric protein assembly, the WAVE-complex, which activates Arp2/3-mediated actin filament nucleation and actin network assembly. A current model of filopodia formation postulates that these structures arise from a dendritic network of lamellipodial actin filaments by selective elongation and bundling. Here, we have analyzed filopodia formation in mammalian cells abrogated in expression of essential components of the lamellipodial actin polymerization machinery. Cells depleted of the WAVE-complex component Nck-associated protein 1 (Nap1), and, in consequence, of lamellipodia, exhibited normal filopodia protrusion. Likewise, the Arp2/3-complex, which is essential for lamellipodia protrusion, is dispensable for filopodia formation. Moreover, genetic disruption of nap1 or the WAVE-orthologue suppressor of cAMP receptor (scar) in Dictyostelium was also ineffective in preventing filopodia protrusion. These data suggest that the molecular mechanism of filopodia formation is conserved throughout evolution from Dictyostelium to mammals and show that lamellipodia and filopodia formation are functionally separable.     

Crystallization and preliminary X-ray crystallographic analysis of strictosidine synthase from Rauvolfia: the first member of a novel enzyme family

Strictosidine synthase is a central enzyme involved in the biosynthesis of almost all plant monoterpenoid indole alkaloids. Strictosidine synthase from Rauvolfia serpentina was heterologously expressed in Escherichia coli. Crystals of the purified recombinant enzyme have been obtained by the hanging-drop technique at 303 K with potassium sodium tartrate tetrahydrate as precipitant. The crystals belong to the space group R3 with cell dimensions of a=b=150.3 A and c=122.4 A. Under cryoconditions (120 K), the crystals diffract to about 2.95 A.     

Human intestinal Caco-2 cells display active transport of benzo[a]pyrene metabolites

Epithelial cells of the gastrointestinal tract are challenged by exposure to many potentially toxic agents including the well-known food contaminant benzo[a]pyrene (B[a]P). They are equipped with a variety of Phase 1- and Phase 2-enzymes that are able to metabolize B[a]P. Furthermore, transmembranous ABC-transport proteins are expressed at the apical pole of these cells. The aim of this study was to investigate whether [14C]B[a]P or products of the metabolism are transported by intestinal cells back into the gut lumen. The intestinal Caco-2 cell line was used as a metabolism and transport model. Experiments with Caco-2 monolayers in the Transwell-system revealed that radiolabeled substance is transported towards the apical (luminal) region. This transport was characterized as active and increased after induction of cytochromes P450 1A1 and 1B1 by beta-naphthoflavone. On the other hand, transport was decreased with the concomitant inhibition of Phase 1-metabolism. TLC-analysis revealed that the primary metabolites of B[a]P found in the supernatant were very polar; other metabolites of less polarity could only be detected in trace amounts. These results indicate that B[a]P is metabolized by Caco-2 cells to highly polar metabolites resulting from biphasic metabolism and that these polar metabolites are subject to an apically directed transport. Chemical inhibition studies showed that P-glycoprotein and MRP1 or 2 were not involved in this polarized B[a]P-metabolite secretion.     

Identification of novel immunodominant CD4+ Th1-type T-cell peptide epitopes from herpes simplex virus glycoprotein D that confer protective immunity

The molecular characterization of the epitope repertoire on herpes simplex virus (HSV) antigens would greatly expand our knowledge of HSV immunity and improve immune interventions against herpesvirus infections. HSV glycoprotein D (gD) is an immunodominant viral coat protein and is considered an excellent vaccine candidate antigen. By using the TEPITOPE prediction algorithm, we have identified and characterized a total of 12 regions within the HSV type 1 (HSV-1) gD bearing potential CD4(+) T-cell epitopes, each 27 to 34 amino acids in length. Immunogenicity studies of the corresponding medium-sized peptides confirmed all previously known gD epitopes and additionally revealed four new immunodominant regions (gD(49-82), gD(146-179), gD(228-257), and gD(332-358)), each containing naturally processed epitopes. These epitopes elicited potent T-cell responses in mice of diverse major histocompatibility complex backgrounds. Each of the four new immunodominant peptide epitopes generated strong CD4(+) Th1 T cells that were biologically active against HSV-1-infected bone marrow-derived dendritic cells. Importantly, immunization of H-2(d) mice with the four newly identified CD4(+) Th1 peptide epitopes but not with four CD4(+) Th2 peptide epitopes induced a robust protective immunity against lethal ocular HSV-1 challenge. These peptide epitopes may prove to be important components of an effective immunoprophylactic strategy against herpes.     

Heterotrimeric G proteins: new tricks for an old dog

Heterotrimeric G proteins are well known for their function in signal transduction downstream of seven transmembrane receptors. More recently, however, genetic analysis in C. elegans and in Drosophila has revealed a second, essential function of these molecules in positioning the mitotic spindle and attaching microtubules to the cell cortex. Five new publications in Cell (Afshar et al., 2004; Du and Macara, 2004 [this issue of Cell]; Hess et al., 2004), Developmental Cell (Martin-McCaffrey et al., 2004), and Current Biology (Couwenbergs et al., 2004) show that this function is conserved in vertebrates and--like the classical pathway--involves cycling of G proteins between GDP and GTP bound conformations.