Carbohydrate carbonyl mobility--the key process in the formation of alpha-dicarbonyl intermediates

Covalently cross-linked proteins are among the major modifications caused by the advanced Maillard reaction. In the present study, the formation pathway of the dideoxyosone N6-(2,3-dihydroxy-5,6-dioxohexyl)-L-lysine is shown. To elucidate the formation of this glucose-derived dideoxyosone D-lactose (O-beta-D-galp-(1-->4)-D-glcp) and D-glucose-6-phosphate were incubated with lysine in the presence of the trapping reagent o-phenylenediamine (OPD). Synthesis and unequivocal structural characterization were reported for the quinoxalines of the dideoxyosones N6-(5,6-dihydroxy-2,3-dioxohexyl)-L-lysine and N6-(2,3-dihydroxy-4,5-dioxohexyl)-L-lysine, respectively. Additionally, dicarbonyl compounds derived from D-erythrose, D-glycero-D-mannoheptose, and D-gluco-L-talooctose were synthesized and structurally characterized.     

The thio-Mitsunobu reaction: a useful tool for the preparation of 2,5-anhydro-2-thio- and 3,5-anhydro-3-thiopentofuranosides

The unprotected methyl L-arabinofuranosides, D-ribofuranosides and D-xylofuranosides are transformed into the corresponding S-acetyl-5-thio derivatives by the thio-Mitsunobu reaction. Mesylation and subsequent reaction with sodium hydrogen carbonate led, depending on the configuration of the intermediate, to 2,5-anhydro-2-thio- or 3,5-anhydro-3-thiopentofuranosides. Due to inversion at C-3 or C-2 during the intramolecular nucleophilic displacement the products exhibit L-lyxo-, D-arabino- or D-lyxo-configuration. Analogously, the methyl 2,3-anhydro-D-ribofuranosides yielded 5-thio-S-acetates with intact 2,3-oxirane groups, which were cyclised with sodium hydrogen carbonate by epoxide ring opening and concomitant ring closure to form exclusively 3,5-anhydro-3-thio-D-xylofuranosides. A related 3,5-anhydro-3-seleno-D-lyxofuranoside was obtained by reaction of a 3,5-di-O-mesyl-D-arabinofuranoside with sodium hydrogen selenide. Several X-ray diffraction analyses proved the structures of the products.     

Ultrastructural characterisation of a nuclear domain highly enriched in survival of motor neuron (SMN) protein

Mutations in the survival of motor neuron (SMN) gene are the major cause of spinal muscular atrophy (SMA). The SMN gene encodes a 38-kDa protein that localises in the cytoplasm and in nuclear bodies termed Gemini of coiled bodies (gems). When visualised by immunofluorescence microscopy, gems often appeared either in close proximity to, or entirely overlapping with coiled (Cajal) bodies (CBs) implying a possible functional relationship between these nuclear domains. With the aim of identifying subnuclear compartments corresponding to gems, we have investigated the intranuclear localisation of SMN and of its interacting protein Gemin2 by immunoelectron microscopy in cultured cells and in liver cells of hibernating dormouse. These antigens are highly enriched in round-shaped electron-dense fibro-granular clusters (EFGCs), which also display a biochemical composition similar to gems visualised by immunofluorescence microscopy. Our data reveal a novel SMN/Gemin2 containing nuclear domain and support the idea that it represents the structural counterpart of gems seen in the light microscope.     

MEK inhibition impairs influenza B virus propagation without emergence of resistant variants

Influenza A and B viruses are still a major worldwide threat. We demonstrate that influenza B virus infection induces signaling via the Raf/MEK/ERK cascade, a process required for efficient virus production. Expression of dominant-negative Raf and ERK mutants or treatment with a MEK inhibitor (U0126) strongly impaired viral propagation, while selective activation of the pathway resulted in increased virus titers. MEK inhibition appears to interfere with a distinct viral nuclear export process. Most importantly, no resistant virus variants emerged in the presence of U0126 demonstrating that influenza viruses cannot easily adapt to the missing cellular function.     

The molecular population genetics of HIV-1 group O

HIV-1 group O originated through cross-species transmission of SIV from chimpanzees to humans and has established a relatively low prevalence in Central Africa. Here, we infer the population genetics and epidemic history of HIV-1 group O from viral gene sequence data and evaluate the effect of variable evolutionary rates and recombination on our estimates. First, model selection tools were used to specify suitable evolutionary and coalescent models for HIV group O. Second, divergence times and population genetic parameters were estimated in a Bayesian framework using Markov chain Monte Carlo sampling, under both strict and relaxed molecular clock methods. Our results date the origin of the group O radiation to around 1920 (1890-1940), a time frame similar to that estimated for HIV-1 group M. However, group O infections, which remain almost wholly restricted to Cameroon, show a slower rate of exponential growth during the twentieth century, explaining their lower current prevalence. To explore the effect of recombination, the Bayesian framework is extended to incorporate multiple unlinked loci. Although recombination can bias estimates of the time to the most recent common ancestor, this effect does not appear to be important for HIV-1 group O. In addition, we show that evolutionary rate estimates for different HIV genes accurately reflect differential selective constraints along the HIV genome.     

NiFe hydrogenase active site biosynthesis: identification of Hyp protein complexes in Ralstonia eutropha

Biosynthesis of the NiFe hydrogenase active site is a complex process involving the action of the Hyp proteins: HypA-HypF. Here we investigate the mechanism of NiFe site biosynthesis in Ralstonia eutropha by examining the interactions between HypC, HypD, HypE, and HypF1. Using an affinity purification procedure based on the Strep-tag II, we purified HypC and HypE from different genetic backgrounds as complexes with other hydrogenase-related proteins and characterized them using immunological analysis. Copurification of HypC and HoxH, the active site-containing subunit of the soluble hydrogenase in R. eutropha, from several different genetic backgrounds suggests that this complex forms early in the maturation process. With respect to the Hyp proteins, it is shown that HypE and HypF1 formed a stable complex both in vivo and in vitro. Furthermore, HypC and HypD functioned as a unit. Together, they were able to interact with HypE to form a range of complexes probably varying in stoichiometry. The HypC/HypD/HypE complexes did not involve HypF1 but appeared to be more stable when HypF1 was also present in the cells. We hypothesize that HypF1 is able to modify some component of the HypC/HypD/HypE complex. Since we have also seen that HypF1 and HypE form a complex, it is likely that HypF1 modifies HypE. On the basis of these results, we propose a complete catalytic cycle for HypE. First, it is modified by HypF1, and then it can form a complex with HypC/HypD. This activated HypE/HypC/HypD complex could then decompose by donating active site components to the immature hydrogenase and regenerate unmodified HypE.     

Dynamics of beta2-adrenergic receptor-ligand complexes on living cells

The agonist-induced dynamic regulation of the beta(2)-adrenergic receptor (beta(2)-AR) on living cells was examined by means of fluorescence correlation spectroscopy (FCS) using a fluorescence-labeled arterenol derivative (Alexa-NA) in hippocampal neurons and in alveolar epithelial type II cell line A549. Alexa-NA specifically bound to the beta(2)-AR of neurons with a K(D) value of 1.29 +/- 0.31 nM and of A549 cells with a K(D) of 5.98 +/- 1.62 nM. The receptor density equaled 4.5 +/- 0.9 microm(-2) in neurons (rho(N)) and 19.9 +/- 2.0 microm(-2) in A549 cells (rho(A549)). Kinetic experiments revealed comparable on-rate constants in both cell types (k(on) = 0.49 +/- 0.03 s(-1) nM(-1) in neurons and k(on) = 0.12 +/- 0.02 s(-1) nM(-1) in A549 cells). In addition to the free ligand diffusing with a D(free) of (2.11 +/- 0.04) x 10(-6) cm(2)/s, in both cell types receptor-ligand complexes with two distinct diffusion coefficients, D(bound1) (fast lateral mobility) and D(bound2) (hindered mobility), were observed [D(bound1) = (5.23 +/- 0.64) x 10(-8) cm(2)/s and D(bound2) = (6.05 +/- 0.23) x 10(-10) cm(2)/s for neurons, and D(bound1) = (2.88 +/- 1.72) x 10(-8) cm(2)/s and D(bound2) = (1.01 +/- 0.46) x 10(-9) cm(2)/s for A549 cells]. Fast lateral mobility of the receptor-ligand complex was detected immediately after addition of the ligand, whereas hindered mobility (D(bound2)) was observed after a delay of 5 min in neurons (up to 38% of total binding) and of 15-20 min in A549 cells (up to 40% of total binding). Thus, the receptor-ligand complexes with low mobility were formed during receptor regulation. Consistently, stimulation of receptor internalization using the adenylate cyclase activator forskolin shifted the ratio of receptor-ligand complexes toward D(bound2). Intracellular FCS measurements and immunocytochemical studies confirmed the appearance of endocytosed receptor-ligand complexes in the cytoplasm subjacent to the plasma membrane after stimulation with the agonist terbutaline (1 microM). This regulatory receptor internalization was blocked after preincubation with propranolol and with a cholesterol-complexing saponin alpha-hederin.     

ERK activation promotes neuronal degeneration predominantly through plasma membrane damage and independently of caspase-3

Our recent studies have shown that extracellular-regulated protein kinase (ERK) promotes cell death in cerebellar granule neurons (CGN) cultured in low potassium. Here we report that the "death" phenotypes of CGN after potassium withdrawal are heterogeneous, allowing the distinction between plasma membrane (PM)-, DNA-, and PM/DNA-damaged populations. These damaged neurons display nuclear condensation that precedes PM or DNA damage. Inhibition of ERK activation either by U0126 or by dominant-negative mitogen-activated protein kinase/ERK kinase (MEK) overexpression results in a dramatic reduction of PM damaged neurons and nuclear condensation. In contrast, overexpression of constitutively active MEK potentiates PM damage and nuclear condensation. ERK-promoted cellular damage is independent of caspase-3. Persistent active ERK translocates to the nucleus, whereas caspase-3 remains in the cytoplasm. Antioxidants that reduced ERK activation and PM damage showed no effect on caspase-3 activation or DNA damage. These data identify ERK as an important executor of neuronal damage involving a caspase-3-independent mechanism.     

The development of the endoparasitoid wasp Venturia canescens in superparasitised Ephestia kuehniella

Using a molecular marker that allows the differentiation of two strains of the solitary endoparasitoid wasp Venturia canescens, the study investigated the influence of host mass and the time interval between ovipositions on the survival and development of larvae from both the first and second laid eggs in superparasitised Ephestia kuehniella. As the time interval between ovipositions increased both overall and superparasitism success decreased, however, time between, and order of, ovipositions had little effect on other developmental parameters. Adult size increased with host mass under both parasitism and superparasitism, while host mortality decreased with host mass under superparasitism. In addition, wasps emerging from superparasitised hosts were larger than wasps from parasitised hosts. The results confirm that for V. canescens on the host E. kuehniella both self- and conspecific-superparasitism will be an adaptive strategy when hosts are the limiting factor.