Differentiation stage-dependent preferred uptake of basolateral (systemic) glutamine into Caco-2 cells results in its accumulation in proteins with a role in cell-cell interaction

Glutamine is an essential amino acid for enterocytes, especially in states of critical illness and injury. In several studies it has been speculated that the beneficial effects of glutamine are dependent on the route of supply (luminal or systemic). The aim of this study was to investigate the relevance of both routes of glutamine delivery to in vitro intestinal cells and to explore the molecular basis for proposed beneficial glutamine effects: (a) by determining the relative uptake of radiolabelled glutamine in Caco-2 cells; (b) by assessing the effect of glutamine on the proteome of Caco-2 cells using a 2D gel electrophoresis approach; and (c) by examining glutamine incorporation into cellular proteins using a new mass spectrometry-based method with stable isotope labelled glutamine. Results of this study show that exogenous glutamine is taken up by Caco-2 cells from both the apical and the basolateral side. Basolateral uptake consistently exceeds apical uptake and this phenomenon is more pronounced in 5-day-differentiated cells than in 15-day-differentiated cells. No effect of exogenous glutamine supply on the proteome was detected. However, we demonstrated that exogenous glutamine is incorporated into newly synthesized proteins and this occurred at a faster rate from basolateral glutamine, which is in line with the uptake rates. Interestingly, a large number of rapidly labelled proteins is involved in establishing cell-cell interactions. In this respect, our data may point to a molecular basis for observed beneficial effects of glutamine on intestinal cells and support results from studies with critically ill patients where parenteral glutamine supplementation is preferred over luminal supplementation.     

Expression of biologically active hordothionins in tobacco. Effects of pre- and pro-sequences at the amino and carboxyl termini of the hordothionin precursor on mature protein expression and sorting

Hordothionins (HTHs) are small anti-bacterial proteins present in barley endosperm which are processed from larger precursor proteins, consisting of an amino-terminal signal peptide (SP), the mature highly basic HTH and a carboxy-terminal acidic peptide (AP). Different HTH precursor proteins were expressed in tobacco to study the effects of the pre-sequences (SP) and pro-sequences (AP) on expression, processing, sorting and biological activity and hence the feasibility of engineering bacterial disease resistance into crops which lack these proteins. Maximum HTH expression levels of approximately 0.7% (11 mol/kg) of total soluble protein in young tobacco leaves were obtained using a semi-synthetic gene construct encoding a complete chimaeric HTH precursor protein. Tenfold lower HTH expression levels (maximum 1.3 mol/kg) were obtained using synthetic gene constructs without the AP-coding sequence and no expression was found in plants containing synthetic HTH gene constructs without SP-and AP-coding sequences. In both cases where expression was found, the precursors were apparently correctly processed, although the HTH produced in plants containing a construct without AP sequence appeared to be slightly modified. No effect on plant phenotype was observed. Localization studies indicated that the HTH was in identical fractions of plants expressing the two different precursors, albeit at a different ratio, and was not secreted into the intercellular spaces of leaves or culture medium by protoplasts. Our results indicated that the AP is not involved in sorting and suggested that it might facilitate transport through membranes. The in vitro toxicity of HTH isolated from transgenic tobacco plants expressing the two different precursor proteins for the bacterial plant pathogen Clavibacter michiganensis subsp. michiganensis appeared similar to that of the HTH purified from barley endosperm.     

Introduction of foreign genes into potato cultivars Bintje and Désirée using an Agrobacterium tumefaciens binary vector

Tuber discs of Solanum tuberosum cv Bintje and Désirée were cocultivated with an Agrobacterium tumefaciens binary vector, carrying both the neomycine phosphotransferase and the E. coli -glucuronidase gene fused to resp. the nopaline synthase and Cauliflower mosaic virus 35S promotor.Inoculated tuber discs produce transgenic shoots in selective media containing kanamycin. The transgenic plants are phenotypically normal and contain the euploid number of chromosomes. Both the neomycin phosphotransferase as well as the -glucuronidase gene are expressed conferring resp. kanamycin resistance and -glucuronidase activity to the plants.Abbreviations GUS -glucuronidase - NPT neomycin phosphotransferase - CaMV Cauliflower Mosaic Virus - BAP 6-benzylaminopurine - GA₃ gibberellic acid - NAA naphthalineacetic acid - LB Luria Broth - MU methylumbelliferone     

Distribution and characterization of plasmid-related sequences in the chromosomal DNA of different thermophilic Methanobacterium strains

The genomes of several thermophilic members of the genus Methanobacterium were analyzed for homology to the related restriction-modification plasmids pFVI and pFZ1 from M. thermoformicicum strains THF and Z-245, respectively. Two plasmid regions, designated FR-I and FR-II, could be identified with chromosomal counterparts in six Methanobacterium strains. Multiple copies of the pFVI-specific element FR-I were detected in the M. thermoformicicum strains CSM3, FF1, FF3 and M. thermoautotrophicum ΔH. Sequence analysis showed that one FR-I element had been integrated in almost identical sequence contexts into the chromosomes of the strains CSM3 and AH. Comparison of the FR-I elements from these strains with that from pFVI revealed that they consisted of two subfragments, boxI (1118 bp) and boxII (383 bp), the order of which is variable. Each subfragment was identical on the sequence level with the corresponding plasmid-borne element and was flanked by terminal direct repeats with the consensus sequence A(A/T)ATTT. These results suggest that FR-I represents a mobile element. FR-II was located on both plasmids pFVI and pFZI, and on the chromosome of M. thermoformicicum strains THF, CSM3 and HN4. Comparison of the nucleotide sequences of the two plasmid FR-II copies and that from the chromosome of strain CSM3 showed that the FR-II segments were approximately 2.5–3.0 kb in size and contained large open reading frames (ORFs) that may encode highly related proteins with an as yet unknown function.     

Early Silurian (Rhuddanian) rugose corals and sponges from the Ak-Kerme Peninsula,Kazakhstan

An assemblage of earliest Silurian (Llandovery, Rhuddanian) fossils from South Kazakhstan (Ak-Kerme Peninsula, Lake Balkhash) contains solitary rugose corals (Calostylis denticulata, Streptelasma? sp., and Cystipaliphyllum sp.) and the demosponge Calycocoelia typicalis, which are described here. This assemblage occurs with previously described brachiopods and constitutes a post extinction survival fauna; such faunas are poorly known and this study fills a gap in our knowledge. All three genera of Rugosa were transitional across the Ordovician–Silurian boundary and are also reported from other parts of Kazakhstan, Uzbekistan, and Tajikistan during the Llandovery. Streptelasma had already been present in Kazakhstan during the Ordovician, while during the Rhuddanian Calostylis immigrated from Baltica or China, and Cystipaliphyllum from the Australian part of Gondwana. Demosponges are rare during the Llandovery but probably had a cosmopolitan distribution. Calycocoelia typicalis marks the first Rhuddanian record of lithistid demosponges, and the first record of Silurian demosponges from Kazakhstan.     

Origin and diversification of the Greater Cape flora: ancient species repository, hot-bed of recent radiation, or both?

Like island-endemic taxa, whose origins are expected to postdate the appearance of the islands on which they occur, biome-endemic taxa should be younger than the biomes to which they are endemic. Accordingly, the ages of biome-endemic lineages may offer insights into biome history. In this study, we used the ages of multiple lineages to explore the origin and diversification of two southern African biomes whose remarkable floristic richness and endemism has identified them as global biodiversity hotspots (succulent karoo and fynbos). We used parsimony optimization to identify succulent karoo- and fynbos-endemic lineages across 17 groups of plants, for which dated phylogenies had been inferred using a relaxed Bayesian (BEAST) approach. All succulent karoo-endemic lineages were less than 17.5 My old, the majority being younger than 10 My. This is largely consistent with suggestions that this biome is the product of recent radiation, probably triggered by climatic deterioration since the late Miocene. In contrast, fynbos-endemic lineages showed a broader age distribution, with some lineages originating in the Oligocene, but most being more recent. Also, in groups having both succulent karoo- and fynbos-endemic lineages, there was a tendency for the latter to be older. These patterns reflect the greater antiquity of fynbos, but also indicate considerable recent speciation, probably through a combination of climatically-induced refugium fragmentation and adaptive radiation.     

Bronchoalveolar lavage fluid from preterm infants with chorioamnionitis inhibits alveolar epithelial repair

Background

Preterm infants are highly susceptible to lung injury. While both chorioamnionitis and antenatal steroids induce lung maturation, chorioamnionitis is also associated with adverse lung development. We investigated the ability of bronchoalveolar lavage fluid (BALF) from ventilated preterm infants to restore alveolar epithelial integrity after injury in vitro, depending on whether or not they were exposed to chorioamnionitis or antenatal steroids. For this purpose, a translational model for alveolar epithelial repair was developed and characterised.

Methods

BALF was added to mechanically wounded monolayers of A549 cells. Wound closure was quantified over time and compared between preterm infants (gestational age < 32 wks) exposed or not exposed to chorioamnionitis and antenatal steroids (≥ 1 dose). Furthermore, keratinocyte growth factor (KGF) and vascular endothelial growth factor (VEGF) were quantified in BALF, and their ability to induce alveolar epithelial repair was evaluated in the model.

Results

On day 0/1, BALF from infants exposed to antenatal steroids significantly increased epithelial repair (40.3 ± 35.5 vs. -6.3 ± 75.0% above control/mg protein), while chorioamnionitis decreased wound-healing capacity of BALF (-2.9 ± 87.1 vs. 40.2 ± 36.9% above control/mg protein). BALF from patients with chorioamnionitis contained less KGF (11 (0-27) vs. 0 (0-4) pg/ml) and less detectable VEGF (66 vs. 95%) on day 0. BALF levels of VEGF and KGF correlated with its ability to induce wound repair. Moreover, KGF stimulated epithelial repair dose-dependently, although the low levels in BALF suggest KGF is not a major modulator of BALF-induced wound repair. VEGF also stimulated alveolar epithelial repair, an effect that was blocked by addition of soluble VEGF receptor-1 (sVEGFr1/Flt-1). However, BALF-induced wound repair was not significantly affected by addition of sVEGFr1.

Conclusion

Antenatal steroids improve the ability of BALF derived from preterm infants to stimulate alveolar epithelial repair in vitro. Conversely, chorioamnionitis is associated with decreased wound-healing capacity of BALF. A definite role for KGF and VEGF in either process could not be established. Decreased ability to induce alveolar epithelial repair after injury may contribute to the association between chorioamnionitis and adverse lung development in mechanically ventilated preterm infants.     

Gli2a protein localization reveals a role for Iguana/DZIP1 in primary ciliogenesis and a dependence of Hedgehog signal transduction on primary cilia in the zebrafish

Background  

In mammalian cells, the integrity of the primary cilium is critical for proper regulation of the Hedgehog (Hh) signal transduction pathway. Whether or not this dependence on the primary cilium is a universal feature of vertebrate Hedgehog signalling has remained contentious due, in part, to the apparent divergence of the intracellular transduction pathway between mammals and teleost fish.     

Stereoselective Binding of Flurbiprofen Enantiomers and their Methyl Esters to Human Serum Albumin Studied by Time-Resolved Phosphorescence

The interaction of the nonsteroidal anti‐inflammatory drug flurbiprofen (FBP) with human serum albumin (HSA) hardly influences the fluorescence of the protein's single tryptophan (Trp). Therefore, in addition to fluorescence, heavy atom‐induced room‐temperature phosphorescence is used to study the stereoselective binding of FBP enantiomers and their methyl esters to HSA. Maximal HSA phosphorescence intensities were obtained at a KI concentration of 0.2 M. The quenching of the Trp phosphorescence by FBP is mainly dynamic and based on Dexter energy transfer. The Stern–Volmer plots based on the phosphorescence lifetimes indicate that (R)‐FBP causes a stronger Trp quenching than (S)‐FBP. For the methyl esters of FBP, the opposite is observed: (S)‐(FBPMe) quenches more than (R)‐FBPMe. The Stern–Volmer plots of (R)‐FBP and (R)‐FBPMe are similar although their high‐affinity binding sites are different. The methylation of (S)‐FBP causes a large change in its effect on the HSA phosphorescence lifetime. Furthermore, the quenching constants of 3.0 × 10⁷ M^?1 s^?1 of the R‐enantiomers and 2.5 × 10⁷ M^?1 s^?1 for the S‐enantiomers are not influenced by the methylation and indicate a stereoselectivity in the accessibility of the HSA Trp to these drugs. Chirality 24:840–846, 2012. © 2012 Wiley Periodicals, Inc.     

Heterosubtypic Neutralizing Monoclonal Antibodies Cross-Protective against H5N1 and H1N1 Recovered from Human IgM+ Memory B Cells

Background

The hemagglutinin (HA) glycoprotein is the principal target of protective humoral immune responses to influenza virus infections but such antibody responses only provide efficient protection against a narrow spectrum of HA antigenic variants within a given virus subtype. Avian influenza viruses such as H5N1 are currently panzootic and pose a pandemic threat. These viruses are antigenically diverse and protective strategies need to cross protect against diverse viral clades. Furthermore, there are 16 different HA subtypes and no certainty the next pandemic will be caused by an H5 subtype, thus it is important to develop prophylactic and therapeutic interventions that provide heterosubtypic protection.

Methods and Findings

Here we describe a panel of 13 monoclonal antibodies (mAbs) recovered from combinatorial display libraries that were constructed from human IgM⁺ memory B cells of recent (seasonal) influenza vaccinees. The mAbs have broad heterosubtypic neutralizing activity against antigenically diverse H1, H2, H5, H6, H8 and H9 influenza subtypes. Restriction to variable heavy chain gene IGHV1-69 in the high affinity mAb panel was associated with binding to a conserved hydrophobic pocket in the stem domain of HA. The most potent antibody (CR6261) was protective in mice when given before and after lethal H5N1 or H1N1 challenge.

Conclusions

The human monoclonal CR6261 described in this study could be developed for use as a broad spectrum agent for prophylaxis or treatment of human or avian influenza infections without prior strain characterization. Moreover, the CR6261 epitope could be applied in targeted vaccine strategies or in the design of novel antivirals. Finally our approach of screening the IgM⁺ memory repertoire could be applied to identify conserved and functionally relevant targets on other rapidly evolving pathogens.