Structural studies on bovine milk galactosyl transferase: effects of pH and denaturants on structure and activity

The pH and denaturant stability of bovine milk galactosyl transferase was studied with particular reference both to aspects of published isolation procedures (acid casein precipitation) and to experiments probing the accessibility of reactive thiol groups. As monitored by catalytic activity or fluorescence spectroscopy, the enzyme undergoes an irreversible inactivation and concomitant structure change below pH 5.0, which is extremely rapid below pH 4.4. At catalytic pH (7.5) the enzyme inactivates (unfolds) at ～ 5 m urea, 2.0 m guanidine hydrochloride and 0.6 mm sodium dodecyl sulphate. The latter detergent apparently binds near Trp residues, as evidenced by a large (>10 nm) blue shift. Extreme caution should be taken in any (acid) casein precipitation steps.     

Toc159- and Toc75-independent import of a transit sequence-less precursor into the inner envelope of chloroplasts

Chloroplast envelope quinone oxidoreductase (ceQORH) is an inner plastid envelope protein that is synthesized without cleavable chloroplast transit sequence for import. In the present work, we studied the in vitro-import characteristics of Arabidopsis ceQORH. We demonstrate that ceQORH import requires ATP and is dependent on proteinaceous receptor components exposed at the outer plastid surface. Competition experiments using small subunit precursor of ribulose-bisphosphate carboxylase/oxygenase and precursor of ferredoxin, as well as antibody blocking experiments, revealed that ceQORH import does not involve the main receptor and translocation channel proteins Toc159 and Toc75, respectively, which operate in import of proteins into the chloroplast. Molecular dissection of the ceQORH amino acid sequence by site-directed mutagenesis and subsequent import experiments in planta and in vitro highlighted that ceQORH consists of different domains that act concertedly in regulating import. Collectively, our results provide unprecedented evidence for the existence of a specific import pathway for transit sequence-less inner plastid envelope membrane proteins into chloroplasts.     

Cytoplasmic utilization of human immunodeficiency virus type 1 genomic RNA is not dependent on a nuclear interaction with gag

In some retroviruses, such as Rous sarcoma virus and prototype foamy virus, Gag proteins are known to shuttle between the nucleus and the cytoplasm and are implicated in nuclear export of the viral genomic unspliced RNA (gRNA) for subsequent encapsidation. A similar function has been proposed for human immunodeficiency virus type 1 (HIV-1) Gag based on the identification of nuclear localization and export signals. However, the ability of HIV-1 Gag to transit through the nucleus has never been confirmed. In addition, the lentiviral Rev protein promotes efficient nuclear gRNA export, and previous reports indicate a cytoplasmic interaction between Gag and gRNA. Therefore, functional effects of HIV-1 Gag on gRNA and its usage were explored. Expression of gag in the absence of Rev was not able to increase cytoplasmic gRNA levels of subgenomic, proviral, or lentiviral vector constructs, and gene expression from genomic reporter plasmids could not be induced by Gag provided in trans. Furthermore, Gag lacking the reported nuclear localization and export signals was still able to mediate an efficient packaging process. Although small amounts of Gag were detectable in the nuclei of transfected cells, a Crm1-dependent nuclear export signal in Gag could not be confirmed. Thus, our study does not provide any evidence for a nuclear function of HIV-1 Gag. The encapsidation process of HIV-1 therefore clearly differs from that of Rous sarcoma virus and prototype foamy virus.     

Informatic search strategies to discover analogues and variants of natural product archetypes

Natural products are a crucial source of antimicrobial agents, but reliance on low-resolution bioactivity-guided approaches has led to diminishing interest in discovery programmes. Here, we demonstrate that two in-house automated informatic platforms can be used to target classes of biologically active natural products, specifically, peptaibols. We demonstrate that mass spectrometry-based informatic approaches can be used to detect natural products with high sensitivity, identifying desired agents present in complex microbial extracts. Using our specialised software packages, we could elaborate specific branches of chemical space, uncovering new variants of trichopolyn and demonstrating a way forward in mining natural products as a valuable source of potential pharmaceutical agents.     

In vivo single-particle imaging of nuclear mRNA export in budding yeast demonstrates an essential role for Mex67p

Many messenger RNA export proteins have been identified; yet the spatial and temporal activities of these proteins and how they determine directionality of messenger ribonucleoprotein (mRNP) complex export from the nucleus remain largely undefined. Here, the bacteriophage PP7 RNA-labeling system was used in Saccharomyces cerevisiae to follow single-particle mRNP export events with high spatial precision and temporal resolution. These data reveal that mRNP export, consisting of nuclear docking, transport, and cytoplasmic release from a nuclear pore complex (NPC), is fast (∼200 ms) and that upon arrival in the cytoplasm, mRNPs are frequently confined near the nuclear envelope. Mex67p functions as the principal mRNP export receptor in budding yeast. In a mex67-5 mutant, delayed cytoplasmic release from NPCs and retrograde transport of mRNPs was observed. This proves an essential role for Mex67p in cytoplasmic mRNP release and directionality of transport.     

Foliar Fatty Acids and Sterols of Soybean Field Fumigated with SO(2)

Sixty-day-old soybean plants were exposed in the field to 78.7 parts per one-hundred million of SO₂ in an open-air fumigation system for 20 days. Leaves from the top one-fourth and bottom one-fourth of the plants were analyzed for chlorophyll, free fatty acids, fatty acid esters, polar lipid fatty acids, and sterols. Fumigated plants had a lower chlorophyll, free fatty acid, and polar lipid content, but a higher fatty acid ester content. Of the individual fatty acids, linoleic and linolenic acid increased with SO₂ fumigation while palmitic acid decreased. SO₂ fumigations had only a minor effect on leaf sterols. In general, the lower, more mature leaves showed a greater response to SO₂ exposure.     

Phospholipase D2 (PLD2) shortens the time required for myeloid leukemic cell differentiation: mechanism of action

Cell differentiation is compromised in acute leukemias. We report that mammalian target of rapamycin (mTOR) and S6 kinase (S6K) are highly expressed in the undifferentiated promyelomonocytic leukemic HL-60 cell line, whereas PLD2 expression is minimal. The expression ratio of PLD2 to mTOR (or to S6K) is gradually inverted upon in vitro induction of differentiation toward the neutrophilic phenotype. We present three ways that profoundly affect the kinetics of differentiation as follows: (i) simultaneous overexpression of mTOR (or S6K), (ii) silencing of mTOR via dsRNA-mediated interference or inhibition with rapamycin, and (iii) PLD2 overexpression. The last two methods shortened the time required for differentiation. By determining how PLD2 participates in cell differentiation, we found that PLD2 interacts with and activates the oncogene Fes/Fps, a protein-tyrosine kinase known to be involved in myeloid cell development. Fes activity is elevated with PLD2 overexpression, phosphatidic acid or phosphatidylinositol bisphosphate. Co-immunoprecipitation indicates a close PLD2-Fes physical interaction that is negated by a Fes-R483K mutant that incapacitates its Src homology 2 domain. All these suggest for the first time the following mechanism: mTOR/S6K down-regulation→PLD2 overexpression→PLD2/Fes association→phosphatidic acid-led activation of Fes kinase→granulocytic differentiation. Differentiation shortening could have a clinical impact on reducing the time of return to normalcy of the white cell counts after chemotherapy in patients with acute promyelocytic leukemia.     

Use of spectral analysis to test hypotheses on the origin of pinnipeds

The evolutionary origin of the pinnipeds (seals, sea lions, and walruses) is still uncertain. Most authors support a hypothesis of a monophyletic origin of the pinnipeds from a caniform carnivore. A minority view suggests a diphyletic origin with true seals being related to the mustelids (otters and ferrets). The phylogenetic relationships of the walrus to other pinniped and carnivore families are also still particularly problematic. Here we examined the relative support for mono- and diphyletic hypotheses using DNA sequence data from the mitochondrial small subunit (12S) rRNA and cytochrome b genes. We first analyzed a small group of taxa representing the three pinniped families (Phocidae, Otariidae, and Odobenidae) and caniform carnivore families thought to be related to them. We inferred phylogenetic reconstructions from DNA sequence data using standard parsimony and neighbor-joining algorithms for phylogenetic inference as well as a new method called spectral analysis (Hendy and Penny) in which phylogenetic information is displayed independently of any selected tree. We identified and compensated for potential sources of error known to lead to selection of incorrect phylogenetic trees. These include sampling error, unequal evolutionary rates on lineages, unequal nucleotide composition among lineages, unequal rates of change at different sites, and inappropriate tree selection criteria. To correct for these errors, we performed additional transformations of the observed substitution patterns in the sequence data, applied more stringent structural constraints to the analyses, and included several additional taxa to help resolve long, unbranched lineages in the tree. We find that there is strong support for a monophyletic origin of the pinnipeds from within the caniform carnivores, close to the bear/raccoon/panda radiation. Evidence for a diphyletic origin was very weak and can be partially attributed to unequal nucleotide compositions among the taxa analyzed. Subsequently, there is slightly more evidence for grouping the walrus with the eared seals versus the true seals. A more conservative interpretation, however, is that the walrus is an early, but not the first, independent divergence from the common pinniped ancestor.      

Protective Efficacy and Immunogenicity of an Adenoviral Vector Vaccine Encoding the Codon-Optimized F Protein of Respiratory Syncytial Virus

Adenoviral vectors (AdV) have received considerable attention for vaccine development because of their high immunogenicity and efficacy. In previous studies, it was shown that DNA immunization of mice with codon-optimized expression plasmids encoding the fusion protein of respiratory syncytial virus (RSV F) resulted in enhanced protection against RSV challenge compared to immunization with plasmids carrying the wild-type cDNA sequence of RSV F. In this study, we constructed AdV carrying the codon-optimized full-length RSV F gene (AdV-F) or the soluble form of the RSV F gene (AdV-Fsol). BALB/c mice were immunized twice with AdV-F or AdV-Fsol and challenged with RSV intranasally. Substantial levels of antibody to RSV F were induced by both AdV vaccines, with peak neutralizing-antibody titers of 1:900. Consistently, the viral loads in lung homogenates and bronchoalveolar lavage fluids were significantly reduced by a factor of more than 60,000. The protection against viral challenge could be measured even 8 months after the booster immunization. AdV-F and AdV-Fsol induced similar levels of immunogenicity and protective efficacy. Therefore, these results encourage further development of AdV vaccines against RSV infection in humans.Human respiratory syncytial virus (RSV) is a highly infectious member of the paramyxovirus family causing upper and lower respiratory tract infections in humans. Serious acute RSV infections, including fatal cases of bronchiolitis and pneumonia, occur particularly in premature infants, immunocompromised adults, and patients with pre-existing chronic lung diseases or underlying heart defects (11, 12, 14, 39, 46, 56). In young children, RSV is the most common respiratory tract pathogen, accounting for approximately 50% of hospitalizations due to lower respiratory tract infections (21). In population-based surveillance studies for hospitalization in Europe, RSV was identified in 42 to 45% of enrolled children younger than 2 years with lower respiratory tract infections, and the rate of hospitalization due to RSV-induced respiratory illnesses was estimated at 3 to 6% among industrialized nations (45). Children with severe RSV infections suffer from oxygen deficiency with cyanosis and require intensive medical care. Furthermore, RSV infection in childhood is suspected to be a risk factor for development of asthma (36, 41, 43, 59). The urgent need for an RSV vaccine is further demonstrated by a study showing that levels of disease burden, mortality, and morbidity caused by RSV infections in the elderly are comparable to those induced by nonpandemic influenza A infections (11). However, the immunization of children with a formalin-inactivated (FI) RSV vaccine in the 1960s resulted in a more severe clinical illness, with two fatal cases, than in nonvaccinated infants following RSV infection, pointing out the difficulties in developing a safe and efficacious RSV vaccine (7, 29). It was shown previously that the enhanced disease severity and the development of pulmonary eosinophilia are mainly attributable to an excessive Th2-polarized immune response (15, 35, 57). Furthermore, the lack of high-affinity antibodies after poor Toll-like receptor stimulation has been suggested to be a key factor of the enhanced disease induced by FI RSV vaccination and subsequent RSV infection shown recently (8). However, the enhanced disease induced by FI RSV could partially be reversed by the chemical reduction of the carbonyl groups produced by prior treatment with aldehyde (34).Passive transfer of a neutralizing monoclonal antibody directed against RSV F (palivizumab) results in significant reduction of hospitalization rate due to RSV infection in children and preterm infants (16, 25), making RSV F a promising vaccine candidate for active immunization. Besides being a target for neutralizing antibodies, RSV F additionally contains cytotoxic-T-cell epitopes (1, 37). Moreover, RSV F based DNA vaccines induced encouraging immune responses of a balanced Th1/Th2 type in mice, as pulmonary eosinophilia and disease-enhancing effects were not observed after viral challenge (4, 5, 19, 31, 52). Additionally, RSV F is highly conserved between the two antigenic subgroups of RSV, which allows generation of cross-reactive antibodies after immunization (26).We recently showed that vaccination with codon-optimized RSV F expression plasmids induced improved humoral immune responses in mice compared to vaccination with wild-type cDNA expression plasmids (52). Consequently, viral load was reduced 13-fold in mice immunized with full-length RSV F and 170-fold in mice immunized with the soluble form of RSV F following RSV challenge in comparison to nonimmunized mice. Based on these results, we inserted the codon-optimized open reading frame (ORF) of both full-length RSV F and its soluble form into a replication-deficient adenoviral serotype 5 vector (AdV), generating AdV-F and AdV-Fsol, respectively, to further enhance the immunogenicity and efficiency of the delivered RSV F transgenes. AdVs were chosen because these viral vectors have been extensively studied and have proven their potential as vaccine vectors in multiple successful preclinical studies (reviewed in references 47, 24, and 51). AdVs are also potent inducers of both humoral and cellular immune responses in animal models and in humans (48, 49, 55). Furthermore, convenience of vector design, ease of handling and a robust antigen expression make AdVs a promising vaccine delivery platform. Another main advantage is their natural tropism for mucosal surfaces, which makes adenoviral vaccines convenient for the purpose of immunization against respiratory pathogens that preferentially initiate infection at the mucosal site (40).However, AdV vaccines expressing the wild-type RSV F protein were tested in several animal models without achieving convincing protection against RSV challenge (13, 22, 23). This might be due to poor RSV F expression levels caused by premature polyadenylation, which could be overcome by codon optimization (53). Hence, here we used the codon-optimized RSV F based AdVs AdV-F and AdV-Fsol and evaluated their potential as RSV vaccines, showing greatly improved vaccine efficacy.