Large-scale production of a therapeutic protein in transgenic tobacco plants: effect of subcellular targeting on quality of a recombinant dog gastric lipase

A recombinant dog gastric lipase with therapeutic potential for the treatment of exocrine pancreatic insufficiency was expressed in transgenic tobacco plants. We targeted the protein using two different signal sequences for either vacuolar retention or secretion. In both cases, an active glycosylated recombinant protein was obtained. The recombinant enzymes and the native enzyme displayed similar properties including acid resistance and acidic optimum pH. The proteolytic maturation and the specific activity of the recombinant proteins, however, were found to be dependent on subcellular compartmentalization. Expression levels of recombinant dog gastric lipase were about 5% and 7% of acid extractable plant proteins for vacuolar retention and secretion respectively. This expression system already has allowed the production of tens of grams of purified lipase through open-field culture of transgenic tobacco plants.     

13C-NMR spectroscopic investigation of α- and β-1,4-D-glucose homooligomers

A complete, unambiguous assignment of all the ¹³C signals of cellobiose and maltose has been achieved using methods such as selective proton decoupling, ¹³C selective spin labeling, and isotopic chemical shift induced by deuterium. The chemical-shift variation of the ¹³C signals with the degree of polymerization in each α or β (1 → 4) series is discussed. The chemical-shift dependence on temperature and solvent in these two series is shown and interpreted in terms of modifications of the solvation and of the conformation.     

The influence of the type of contraction on the masseter muscle EMG power spectrum

Different behaviours of the EMG power spectrum across increasing force levels have been reported for the masseter muscle. A factor that could explain these different behaviours may be the type of contraction used, as was recently shown for certain upper limb muscles⁵. The purpose of this study was to compare, between two types of isometric contractions, the behaviour of EMG power spectrum statistics (median frequency (MF) and mean power frequency (MPF)) obtained across increasing force levels. Ten women exerted, while biting in the intercuspal position, three 5 s ramp contractions that increased linearly from 0 to 100% of the maximal voluntary contraction (MVC). They also completed three step contractions (constant EMG amplitude) at each of the following levels: 20, 40, 60 and 80% MVC. EMG signals from the masseter muscle were recorded with miniature surface electrodes. The RMS, as well as the MPF and MF of the power spectrum were calculated at 20, 40, 60 and 80% MVC for each type of contraction. As expected, the RMS values showed similar increases with increasing levels of effort for both types of contractions. Different behaviours for both MPF (contraction^*force interaction, ANOVA, P<0.05) and MF (contraction^*force interaction, ANOVA, P>0.05) across increasing levels of effort were found between the two types of contraction. The use of step contractions gave rise to a decrease of both MPF and MF with increasing force, while the use of ramp contractions gave rise to an increase in both statistics up to at least 40% MVC followed by a decrease at higher force levels. These findings suggest that the type of contraction used does influence the behaviour of the spectral statistics across increasing force levels and that this could explain the differences obtained in previous studies for the masseter muscle.     

Parallel Exploitation of Diverse Host Nutrients Enhances Salmonella Virulence

Pathogen access to host nutrients in infected tissues is fundamental for pathogen growth and virulence, disease progression, and infection control. However, our understanding of this crucial process is still rather limited because of experimental and conceptual challenges. Here, we used proteomics, microbial genetics, competitive infections, and computational approaches to obtain a comprehensive overview of Salmonella nutrition and growth in a mouse typhoid fever model. The data revealed that Salmonella accessed an unexpectedly diverse set of at least 31 different host nutrients in infected tissues but the individual nutrients were available in only scarce amounts. Salmonella adapted to this situation by expressing versatile catabolic pathways to simultaneously exploit multiple host nutrients. A genome-scale computational model of Salmonella in vivo metabolism based on these data was fully consistent with independent large-scale experimental data on Salmonella enzyme quantities, and correctly predicted 92% of 738 reported experimental mutant virulence phenotypes, suggesting that our analysis provided a comprehensive overview of host nutrient supply, Salmonella metabolism, and Salmonella growth during infection. Comparison of metabolic networks of other pathogens suggested that complex host/pathogen nutritional interfaces are a common feature underlying many infectious diseases.     

Flagellin/TLR5 signalling activates renal collecting duct cells and facilitates invasion and cellular translocation of uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) colonizing kidneys is the main cause of acute pyelonephritis. TLR5 that senses flagellin was shown to be highly expressed in the bladder and to participate in host defence against flagellated UPEC, although its role in kidneys still remains elusive. Here we show that TLR5 is expressed in renal medullary collecting duct (MCD) cells, which represent a preferential site of UPEC adhesion. Flagellin, like lipopolysaccharide, stimulated the production of the chemoattractant chemokines CXCL1 and CXCL2, and subsequent migration capacity of neutrophils in cultured wild‐type (WT) and Tlr4^?/? MCDs, but not in Tlr5^?/? MCDs. UPEC can translocate across intact MCD layers without altering tight junctions. Strikingly, the invasion capacity and transcellular translocation of the UPEC strain HT7 were significantly lower in Tlr5^?/? than in WT MCDs. The non‐motile HT7ΔfliC mutant lacking flagellin also exhibited much lower translocation capacities than the HT7 isolates. Finally, Tlr5^?/? kidneys exhibited less infiltrating neutrophils than WT kidneys one day after the transurethral inoculation of HT7, and greater delayed renal bacterial loads in the day 4 post‐infected Tlr5^?/? kidneys. Overall, these findings indicate that the epithelial TLR5 participates to renal antibacterial defence, but paradoxically favours the translocation of UPEC across intact MCD cell layers.     

Low-temperature electron transfer in photosystem II: a tyrosyl radical and semiquinone charge pair

The states induced by illumination at 7 K in the oxygen-evolving enzyme (PSII) from Thermosynechococcus elongatus were studied by EPR. In the S(0) and S(1) redox states, two g approximately 2 EPR signals, a split signal and a g = 2.03 signal, respectively, were generated by illumination with visible light. These signals were comparable to those already reported in plant PSII in terms of their g value, shape, and stability at low temperatures. We report that the formation and decay of these signals correlate with EPR signals from the semiquinone of the first quinone electron acceptor, Q(A)(-). The light-induced EPR signals from oxidized side-path electron donors (Cyt b(559), Car, and Chl(Z)) were also measured, and from these and the signals from Q(A)(-), estimates were made of the proportion of centers involved in the formation of the g approximately 2 signals (approximately 50% in S(0) and 40% in S(1)). Comparisons with the signals generated in plant PSII indicated approximately similar yields for the S(0) split signal. A single laser flash at 7 K induced more than 75% of the maximum split and g = 2.03 EPR signal observed by continuous illumination, with no detectable oxidation of side-path donors. The matching electron acceptor side reactions, the high quantum yield, and the relatively large proportion of centers involved support earlier suggestions that the state being monitored is Tyr(Z)(*)Q(A)(-), with the g approximately 2 EPR signals arising from Tyr(Z)(*) interacting magnetically with the Mn complex. The current picture of the photochemical reactions occurring in PSII at low temperatures is reassessed.     

Marketing approval of mogamulizumab: A triumph for glyco-engineering

Therapeutic properties of antibodies strongly depend on the composition of their glycans. Most of the currently approved antibodies are produced in mammalian cell lines, which yield mixtures of different glycoforms that are close to those of humans, but not fully identical. Glyco-engineering is being developed as a method to control the composition of carbohydrates and to enhance the pharmacological properties of mAbs. The recent approval in Japan of mogamulizumab (POTELIGEO^®), the first glyco-engineered antibody to reach the market, is a landmark in the field of therapeutic antibodies. Mogamulizumab is a humanized mAb derived from Kyowa Hakko Kirin’s POTELLIGENT^® technology, which produces antibodies with enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity. The approval was granted April 30, 2012 by the Japanese Ministry of Health, Labour and Welfare for patients with relapsed or refractory CCR4-positive adult T-cell leukemia-lymphoma.     

Impact of Urban Pollution from the Hanoi Area on Benthic Diatom Communities Collected from the Red, Nhue and Tolich Rivers (Vietnam)

The effects of urban pollution from Hanoi city on the benthic diatom communities of the Nhue–Tolich river system were studied during the 2003 dry season. Benthic diatoms were allowed to grow on glass slides suspended in the water flow for 4 weeks. To reveal the relationship between water quality and diatom communities, Canonical correspondence analysis (CCA) was used on data concerning relative abundances of diatom species and environmental variables. Two diatom indices, IPS and DAIpo, were applied to evaluate water quality in the three rivers. A total of 291 diatom taxa were found in the Red, Nhue and Tolich Rivers. These were mainly cosmopolitan taxa, with some tropical, subtropical and endemic taxa. The most abundant taxa at the Red site were Aulacoseira granulata, Achnanthidium minutissimum, Encyonema minutum, Navicula recens and other halophilous taxa such as Nitzschia kurzii, Seminavis strigosa, Entomoneis paludosa, Bacillaria paradoxa. Diatom assemblages at the Tolich site consisted mainly of Nitzschia umbonata, Nitzschia palea and Eolimna minima. Diatom density ranged from 660 to 30,000 cells/cm². Environmental variables and diatom assemblage composition at all sites were significantly correlated. Two diatom indices gave similar results and indicate the Tolich River with the lowest values as a highly polluted site.