Ecotype diversity in the marine picoeukaryote Ostreococcus (Chlorophyta, Prasinophyceae)

The importance of the cyanobacteria Prochlorococcus and Synechococcus in marine ecosystems in terms of abundance and primary production can be partially explained by ecotypic differentiation. Despite the dominance of eukaryotes within photosynthetic picoplankton in many areas a similar differentiation has never been evidenced for these organisms. Here we report distinct genetic [rDNA 18S and internal transcribed spacer (ITS) sequencing], karyotypic (pulsed-field gel electrophoresis), phenotypic (pigment composition) and physiological (light-limited growth rates) traits in 12 Ostreococcus strains (Prasinophyceae) isolated from various marine environments and depths, which suggest that the concept of ecotype could also be valid for eukaryotes. Internal transcribed spacer phylogeny grouped together four deep strains isolated between 90 m and 120 m depth from different geographical origins. Three deep strains displayed larger chromosomal bands, different chromosome hybridization patterns, and an additional chlorophyll (chl) c-like pigment. Furthermore, growth rates of deep strains show severe photo-inhibition at high light intensities, while surface strains do not grow at the lowest light intensities. These features strongly suggest distinct adaptation to environmental conditions encountered at surface and the bottom of the oceanic euphotic zone, reminiscent of that described in prokaryotes.     

Adult-derived stem cells and their potential for use in tissue repair and molecular medicine

This report reviews three categories of precursor cells present within adults. The first category of precursor cell, the epiblast-like stem cell, has the potential of forming cells from all three embryonic germ layer lineages, e.g., ectoderm, mesoderm, and endoderm. The second category of precursor cell, the germ layer lineage stem cell, consists of three separate cells. Each of the three cells is committed to form cells limited to a specific embryonic germ layer lineage. Thus the second category consists of germ layer lineage ectodermal stem cells, germ layer lineage mesodermal stem cells, and germ layer lineage endodermal stem cells. The third category of precursor cells, progenitor cells, contains a multitude of cells. These cells are committed to form specific cell and tissue types and are the immediate precursors to the differentiated cells and tissues of the adult. The three categories of precursor cells can be readily isolated from adult tissues. They can be distinguished from each other based on their size, growth in cell culture, expressed genes, cell surface markers, and potential for differentiation. This report also discusses new findings. These findings include the karyotypic analysis of germ layer lineage stem cells; the appearance of dopaminergic neurons after implantation of naive adult pluripotent stem cells into a 6-hydroxydopamine-lesioned Parkinson's model; and the use of adult stem cells as transport mechanisms for exogenous genetic material. We conclude by discussing the potential roles of adult-derived precursor cells as building blocks for tissue repair and as delivery vehicles for molecular medicine.     

Controlling {beta}-amyloid oligomerization by the use of naphthalene sulfonates: trapping low molecular weight oligomeric species

Aggregation of proteins and peptides has been shown to be responsible for several diseases known as amyloidoses, which include Alzheimer disease (AD), prion diseases, among several others. AD is a neurodegenerative disorder caused primarily by the aggregation of beta-amyloid peptide (Abeta). Here we describe the stabilization of small oligomers of Abeta by the use of sulfonated hydrophobic molecules such as AMNS (1-amino-5-naphthalene sulfonate); 1,8-ANS (1-anilinonaphthalene-8-sulfonate) and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate). The experiments were performed with either Abeta-1-42 or with Abeta-13-23, a shorter version of Abeta that is still able to form amyloid fibrils in vitro and contains amino acid residues 16-20, previously shown to be essential to peptide-peptide interaction and fibril formation. All sulfonated molecules tested were able to prevent Abeta aggregation in a concentration dependent fashion in the following order of efficacy: 1,8-ANS < AMNS < bis-ANS. Size exclusion chromatography revealed that in the presence of bis-ANS, Abeta forms a heterogeneous population of low molecular weight species that proved to be toxic to cell cultures. Since the ANS compounds all have apolar rings and negative charges (sulfonate groups), both hydrophobic and electrostatic interactions may contribute to interpeptide contacts that lead to aggregation. We also performed NMR experiments to investigate the structure of Abeta-13-23 in SDS micelles and found features of an alpha-helix from Lys(16) to Phe(20). 1H TOCSY spectra of Abeta-13-23 in the presence of AMNS displayed a chemical-shift dispersion quite similar to that observed in SDS, which suggests that in the presence of AMNS this peptide might adopt a conformation similar to that reported in the presence of SDS. Taken together, our studies provide evidence for the crucial role of small oligomers and their stabilization by sulfonate hydrophobic compounds.     

Ca2+-calmodulin-dependent facilitation and Ca2+ inactivation of Ca2+ release-activated Ca2+ channels

In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. Here, we report that intracellular Ca2+ modulates CRAC channel activity through both positive and negative feedback steps in RBL-1 cells. Under conditions in which cytoplasmic Ca2+ concentration can fluctuate freely, we find that store-operated Ca2+ entry is impaired either following overexpression of a dominant negative calmodulin mutant or following whole-cell dialysis with a calmodulin inhibitory peptide. The peptide had no inhibitory effect when intracellular Ca2+ was buffered strongly at low levels. Hence, Ca2+-calmodulin is not required for the activation of CRAC channels per se but is an important regulator under physiological conditions. We also find that the plasma membrane Ca2+ATPase is the dominant Ca2+ efflux pathway in these cells. Although the activity of the Ca2+ pump is regulated by calmodulin, the store-operated Ca2+ entry is more sensitive to inhibition by the calmodulin mutant than by Ca2+ extrusion. Hence, these two plasmalemmal Ca2+ transport systems may differ in their sensitivities to endogenous calmodulin. Following the activation of Ca2+ entry, the rise in intracellular Ca2+ subsequently feeds back to further inhibit Ca2+ influx. This slow inactivation can be activated by a relatively brief Ca2+ influx (30-60 s); it reverses slowly and is not altered by overexpression of the calmodulin mutant. Hence, the same messenger, intracellular Ca2+, can both facilitate and inactivate Ca2+ entry through store-operated CRAC channels and through different mechanisms.     

Mutations within the programmed cell death 10 gene cause cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are hamartomatous vascular malformations characterized by abnormally enlarged capillary cavities without intervening brain parenchyma. They cause seizures and cerebral hemorrhages, which can result in focal neurological deficits. Three CCM loci have been mapped, and loss-of-function mutations were identified in the KRIT1 (CCM1) and MGC4607 (CCM2) genes. We report herein the identification of PDCD10 (programmed cell death 10) as the CCM3 gene. The CCM3 locus has been previously mapped to 3q26-27 within a 22-cM interval that is bracketed by D3S1763 and D3S1262. We hypothesized that genomic deletions might occur at the CCM3 locus, as reported previously to occur at the CCM2 locus. Through high-density microsatellite genotyping of 20 families, we identified, in one family, null alleles that resulted from a deletion within a 4-Mb interval flanked by markers D3S3668 and D3S1614. This de novo deletion encompassed D3S1763, which strongly suggests that the CCM3 gene lies within a 970-kb region bracketed by D3S1763 and D3S1614. Six additional distinct deleterious mutations within PDCD10, one of the five known genes mapped within this interval, were identified in seven families. Three of these mutations were nonsense mutations, and two led to an aberrant splicing of exon 9, with a frameshift and a longer open reading frame within exon 10. The last of the six mutations led to an aberrant splicing of exon 5, without frameshift. Three of these mutations occurred de novo. All of them cosegregated with the disease in the families and were not observed in 200 control chromosomes. PDCD10, also called "TFAR15," had been initially identified through a screening for genes differentially expressed during the induction of apoptosis in the TF-1 premyeloid cell line. It is highly conserved in both vertebrates and invertebrates. Its implication in cerebral cavernous malformations strongly suggests that it is a new player in vascular morphogenesis and/or remodeling.     

Serendipitous identification of natural Intergenotypic recombinants of hepatitis C in Ireland

Background

Encephalitis caused by flaviviruses, Japanese encephalitis virus (JEV) and West Nile virus (WNV) is responsible for significant morbidity and mortality in many endemic countries. Dengue-2 (Den-2) virus is a recent addition to the list of encephalitogenic viruses, after its Central Nervous System (CNS) invasion capability has been established. There is a wide array of laboratory tools that have helped us not only in the diagnosis of these conditions but also in understanding their pathogenesis and pathology. However, there are no reports of Shell Vial Culture (SVC), a centrifuge enhanced tissue culture assay that has revolutionized viral culturing in terms of rapidity and sensitivity being optimized for these flaviviral encephalitic conditions. The present study is an attempt to standardize and evaluate the usefulness of SVC for the laboratory diagnosis of JE, WN and Den-2 encephalitis cases and to compare it with Indirect Immunofluorescence (IIF) technique that detects cell associated virus antigen. Analysis of the various clinical parameters with respect to viral etiology has also been carried out.

Results

Pediatric patients constituted the major group involved in the study (92%). Etiological diagnosis of viral encephalitis could be established in twenty nine (58%) patients. JE encephalitis was the commonest with 19 (39%) cases being positive followed by, WN (9 cases-18%) and Den-2 (one case). IIF test could detect antigens of JE, WN and Den-2 viruses in 16(32%), 7(14%) and 1 case respectively. Shell vial culture assay picked up all cases that were positive by IIF test. In addition, SVC assay could detect 3 and 2 more cases of JE and WN encephalitis respectively, that were negative by the IIF test.

Conclusion

Shell vial culture is a rapid and efficient tool for the etiological diagnosis of JE, WN and Den-2 encephalitis cases. Early, prompt collection, transport and processing of the CSF samples, would make SVC a better method for the rapid diagnosis of these flaviviral infections.     

Variability of the microbial community in the western Antarctic Peninsula from late fall to spring during a low ice cover year

Although winter conditions play a major role in determining the productivity of the western Antarctic Peninsula (WAP) waters for the following spring and summer, a few studies have dealt with the seasonal variability of microorganisms in the WAP in winter. Moreover, because of regional warming, sea-ice retreat is happening earlier in spring, at the onset of the production season. In this context, this study describes the dynamics of the marine microbial community in the Melchior Archipelago (WAP) from fall to spring 2006. Samples were collected monthly to biweekly at four depths from the surface to the aphotic layer. The abundance and carbon content of bacteria, phytoplankton and microzooplankton were analyzed using flow cytometry and inverted microscopy, and bacterial richness was examined by PCR–DGGE. As expected, due to the extreme environmental conditions, the microbial community abundance and biomass were low in fall and winter. Bacterial abundance ranged from 1.2 to 2.8 × 10⁵ cells ml^?1 showing a slight increase in spring. Phytoplankton biomass was low and dominated by small cells (<2 μm) in fall and winter (average chlorophyll a concentration, Chl-a, of, respectively, 0.3 and 0.13 μg l^?1). Phytoplankton biomass increased in spring (Chl-a up to 1.13 μg l^?1), and, despite potentially adequate growth conditions, this rise was small and phytoplankton was still dominated by small cells (2–20 μm). In addition, the early disappearing of sea-ice in spring 2006 let the surface water exposed to ultraviolet B radiations (UVBR, 280–320 nm), which seemed to have a negative impact on the microbial community in surface waters.     

A Simple Method for the Reconstitution of Membrane Proteins into Giant Unilamellar Vesicles

A simple method for the reconstitution of membrane protein from submicron proteoliposomes into giant unilamellar vesicles (GUVs) is presented here: This method does not require detergents, fusion peptides or a dehydration step of the membrane protein solution. In a first step, GUVs of lipids were formed by electroformation, purified and concentrated; and in a second step, the concentrated GUV solution was added to a small volume of vesicles or proteoliposomes. Material transfer from submicron vesicles and proteoliposomes to GUVs occurred spontaneously and was characterized with fluorescent microscopy and patch-clamp recordings. As a functional test, the voltage-dependent, anion-selective channel protein was reconstituted into GUVs, and its electrophysiological activity was monitored with the patch clamp. This method is versatile since it is independent of the presence of the protein, as demonstrated by the fusion of fluorescently labeled submicron vesicles and proteoliposomes with GUVs.     

The Role of eIF1 in Translation Initiation Codon Selection in Caenorhabditis elegans

The selection of a proper AUG start codon requires the base-pairing interactions between the codon on the mRNA and the anticodon of the initiator tRNA. This selection process occurs in a pre-initiation complex that includes multiple translation initiation factors and the small ribosomal subunit. To study how these initiation factors are involved in start codon recognition in multicellular organisms, we isolated mutants that allow the expression of a GFP reporter containing a non-AUG start codon. Here we describe the characterization of mutations in eif-1, which encodes the Caenorhabditis elegans translation initiation factor 1 (eIF1). Two mutations were identified, both of which are substitutions of amino acid residues that are identical in all eukaryotic eIF1 proteins. These residues are located in a structural region where the amino acid residues affected by the Saccharomyces cerevisiae eIF1 mutations are also localized. Both C. elegans mutations are dominant in conferring a non-AUG translation initiation phenotype and lead to growth arrest defects in homozygous animals. By assaying reporter constructs that have base changes at the AUG start codon, these mutants are found to allow expression from most reporters that carry single base changes within the AUG codon. This trend of non-AUG mediated initiation was also observed previously for C. elegans eIF2β mutants, indicating that these two factors play a similar role. These results support that eIF1 functions in ensuring the fidelity of AUG start codon recognition in a multicellular organism.TRANSLATION initiation is thought to be one of the most complex cellular processes in eukaryotes. It involves at least 12 translation initiation factors (eIFs) comprising over 30 polypeptides (Pestova et al. 2007). These factors bring together an initiator methionyl tRNA (Met-tRNAi), the small ribosomal subunit, and a mRNA to form a 48S initiation complex. An important role performed by this complex is to select an AUG codon to initiate translation of the mRNA. Since the first AUG at the 5′ end of most mRNAs is selected as the start site, it is believed that the initiation complex scans for an AUG start codon as it moves from the 5′-capped end of the mRNA toward the 3′ end, as proposed in the ribosomal scanning model (Kozak 1978; Kozak 1989). The recognition of the AUG start codon is mediated by the anticodon of the Met-tRNAi, and the matching base-pairing interactions between the codon of the mRNA and the anticodon determine the site of initiation (Cigan et al. 1988). These base-pairing interactions are essential, but are likely not the only components required for accurately selecting the correct AUG start codon. Numerous initiation factors along with base-pairing interactions have been shown to aid in the AUG recognition process (Pestova et al. 2007).Translation initiation factors involved in start codon selection fidelity were first identified through genetic studies performed in the yeast Saccharomyces cerevisiae. Mutant strains with a modified His4 gene that had an AUU instead of an AUG at the native start site were selected for the ability to survive on media lacking histidine (Donahue et al. 1988; Castilho-Valavicius et al. 1990). These mutants were found to be able to produce the His4 protein by using a downstream inframe UUG codon (the third codon within the His4 coding region) as the translation start site. Further analyses determined that non-AUG initiation occurred mostly from a UUG codon and not significantly from other codons (Huang et al. 1997). These mutants defined five genetic loci and were named sui1-sui5 (suppressor of initiation codon) on the basis of their ability to initiate translation at a non-AUG codon.The sui1 suppressors were found to have missense mutations in eIF1. These missense mutations showed semidominant or codominant properties in non-AUG translation initiation while deletion of the eIF1 gene led to lethality in yeast (Yoon and Donahue 1992). eIF1 is a highly conserved protein with a size of approximately 12 kDa that plays a vital role in multiple translation initiation steps. eIF1 is incorporated into a multifactor complex that includes eIF1A, eIF3, and eIF5 and stimulates the recruiting of the ternary complex (consisting of eIF2 · GTP and the charged Met-tRNAi) to the small ribosomal subunit to form the 43S pre-initiation complex (Singh et al. 2004). eIF1 acts synergistically with eIF1A to promote continuous ribosomal scanning for AUG codons by stabilizing an open conformation that allows mRNA to pass through the complex (Maag et al. 2005; Cheung et al. 2007; Passmore et al. 2007). It also mediates the assembly of the ribosomal initiation complex at the AUG start codon (Pestova et al. 1998). eIF1 dissociates from the complex upon recognition of the AUG codon and this dissociation is necessary to trigger a series of conformational changes leading to the translation elongation phase (Algire et al. 2005). Consistent with these roles, sui1 mutations reduce the affinity of eIF1 for the ribosome and cause premature release of eIF1 at non-AUG codons (Cheung et al. 2007). Other sui mutations support the involvement of four additional genes in translation initiation fidelity in yeast. Mutations have been isolated in the heterotrimeric eIF2 as SUI2 (α-subunit) (Cigan et al. 1989), SUI3 (β-subunit) (Donahue et al. 1988), and SUI4 (γ-subunit) (Huang et al. 1997), and a mutation in eIF5 corresponds to the SUI5 mutant (Huang et al. 1997).However, the genetic studies that identified these translation fidelity mutants were conducted only in yeast. It is not known if there are similar mechanisms regulating translation initiation fidelity in multicellular organisms. To address this question, we designed a genetic system to isolate C. elegans mutants that have reduced fidelity in AUG start codon selection (Zhang and Maduzia 2010). Mutants were selected on the basis of their ability to express a GFP reporter that contains a GUG codon in place of its native translation start site. Here we report the characterization of two mutants that have mutations in eIF1. Unlike yeast sui1 mutants, which preferred the UUG codon, these mutants are capable of using a subset of non-AUG codons for translation initiation. Our results are consistent with eIF1 playing a role in the fidelity of AUG codon selection, perhaps by discriminating base-pairing interactions between the codon and anticodon during start-site selection.