Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Substrates for Human Embryonic Stem Cell Maintenance

Human embryonic stem cells (hESCs) are pluripotent cells that have indefinite replicative potential and the ability to differentiate into derivatives of all three germ layers. hESCs are conventionally grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) or feeder cells of human origin. In addition, feeder-free culture systems can be used to support hESCs, in which the adhesive substrate plays a key role in the regulation of stem cell self-renewal or differentiation. Extracellular matrix (ECM) components define the microenvironment of the niche for many types of stem cells, but their role in the maintenance of hESCs remains poorly understood. We used a proteomic approach to characterize in detail the composition and interaction networks of ECMs that support the growth of self-renewing hESCs. Whereas many ECM components were produced by supportive and unsupportive MEF and human placental stromal fibroblast feeder cells, some proteins were only expressed in supportive ECM, suggestive of a role in the maintenance of pluripotency. We show that identified candidate molecules can support attachment and self-renewal of hESCs alone (fibrillin-1) or in combination with fibronectin (perlecan, fibulin-2), in the absence of feeder cells. Together, these data highlight the importance of specific ECM interactions in the regulation of hESC phenotype and provide a resource for future studies of hESC self-renewal.     

The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine in Arabidopsis

Despite its importance in a variety of plant defense responses, our understanding of how jasmonic acid (JA) functions at the biochemical level is limited. Several amino acid conjugates of JA were tested for their ability to complement the JA-insensitive Arabidopsis thaliana mutant jar1-1. Unlike free JA, JA-Ile inhibited root growth in jar1-1 to the same extent as in the wild type, whereas JA-Val, JA-Leu, and JA-Phe were ineffective inhibitors in both genotypes. Thin-layer chromatography and gas chromatography-mass spectrometry (GC-MS) analysis of products produced in vitro by recombinant JAR1 demonstrated that this enzyme forms JA-amido conjugates with several amino acids, including JA-Ile. JA-Val, -Leu, -Ile, and -Phe were each quantified in Arabidopsis seedlings by GC-MS. JA-Ile was found at 29.6 pmole g(-1) fresh weight (FW) in the wild type but was more than sevenfold lower in two jar1 alleles. JA-Leu, -Val, and -Phe were present at only low levels in both genotypes. Expression of wild-type JAR1 in transgenic jar1-1 plants restored sensitivity to JA and elevated JA-Ile to the same level as in the wild type. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) conjugated to JA was also found in plant tissue at 18.4 pmole g(-1) FW. JA-ACC was determined not be an effective jasmonate root inhibitor, and surprisingly, was twofold higher in the mutants than in the wild type. This suggests that another JA-conjugating enzyme(s) is present in Arabidopsis. Synthesis of JA-ACC might provide a mechanism to coregulate the availability of JA and ACC for conversion to the active hormones JA-Ile and ethylene, respectively. We conclude that JAR1 is a JA-amino synthetase that is required to activate JA for optimal signaling in Arabidopsis. Plant hormone activation by conjugation to amino acids and the enzymes involved in their formation were previously unknown.     

A case of Lemierre's syndrome following Epstein-Barr virus infection

Lemierre's syndrome, or necrobacillosis, is a life-threatening septic thrombophlebitis of the internal jugular vein. The causative organism is Fusobacterium necrophorum. Here we report a case of Lemierre's syndrome in a young male and describe the clinical presentations and treatment. Epstein-Barr virus (EBV) was a suspected predisposing factor in this case.     

Human mitochondrial thymidine kinase is coded for by a gene on chromosome 16 of the nucleus

The expression of human mitochondrial thymidine kinase (mt TK) was investigated by polyacrylamide electrophoresis in 19 independent human-mouse somatic cell hybrids which allowed all human chromosomes to be analyzed. In 8 hybrid clones the presence of this enzymatic activity could be demonstrated. Human mt TK segregated concordantly with human adenine phosphoribosyltransferase (APRT) and human chromosome 16. Discordant segregation with all other human chromosomes was demonstrated by karyotype and isozyme analyses. These results suggest that human mt TK is coded for by a gene on chromosome 16 of the nucleus. Thus human mt TK is genetically different from human cytosol thymidine kinase which is coded for by a gene on chromosome 17. The appearance of one heteropolymer band after electrophoretic separation of human and murine mt TK supports the notion that both enzymes have dimeric structures.     

Rab6 interacts with the mint3 adaptor protein

The Rab6 GTPase regulates a retrograde transport route connecting endosomes and the endoplasmic reticulum (ER) via the Golgi apparatus. Recently it was shown that active (GTP-loaded) Rab6A regulates intracellular processing of the amyloid precursor protein (APP). To characterize the role of Rab6A in APP trafficking and to identify effector proteins of the active Rab6A protein, we screened a human placenta cDNA library using the yeast two-hybrid system. We isolated an interacting cDNA clone encoding part of the adaptor protein mint3. The interaction between Rab6A and mint3 is GTP-dependent and requires the complete phosphotyrosine-binding (PTB) domain of the mint protein, which also mediates the association with APP. By confocal microscopy we show that Rab6A, mint3 and APP co-localize at Golgi membranes in HeLa cells. Density gradient centrifugation of cytosolic extracts confirms a common distribution of these three proteins. Our data suggest that mint3 links Rab6A to APP traffic.     

A Multiplex PCR for the Simultaneous Detection and Genotyping of the Echinococcus granulosus Complex

Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1–G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1–G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6–G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.