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.     

A Monoacylglycerol Lipase from Mycobacterium smegmatis Involved in Bacterial Cell Interaction

MSMEG_0220 from Mycobacterium smegmatis, the ortholog of the Rv0183 gene from M. tuberculosis, recently identified and characterized as encoding a monoacylglycerol lipase, was cloned and expressed in Escherichia coli. The recombinant protein (rMSMEG_0220), which exhibits 68% amino acid sequence identity with Rv0183, showed the same substrate specificity and similar patterns of pH-dependent activity and stability as the M. tuberculosis enzyme. rMSMEG_0220 was found to hydrolyze long-chain monoacylglycerol with a specific activity of 143 ± 6 U mg⁻¹. Like Rv0183 in M. tuberculosis, MSMEG_0220 was found to be located in the cell wall. To assess the in vivo role of the homologous proteins, an MSMEG_0220 disrupted mutant of M. smegmatis (MsΔ0220) was produced. An intriguing change in the colony morphology and in the cell interaction, which were partly restored in the complemented mutant containing either an active (ComMsΔ0220) or an inactive (ComMsΔ0220S111A) enzyme, was observed. Growth studies performed in media supplemented with monoolein showed that the ability of both MsΔ0220 and ComMsΔ0220S111A to grow in the presence of this lipid was impaired. Moreover, studies of the antimicrobial susceptibility of the MsΔ0220 strain showed that this mutant is more sensitive to rifampin and more resistant to isoniazid than the wild-type strain, pointing to a critical structural role of this enzyme in mycobacterial physiology, in addition to its function in the hydrolysis of exogenous lipids.Tuberculosis, which is caused by Mycobacterium tuberculosis, is a major public health issue worldwide. Because of the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains and the high incidence of HIV and tuberculosis coinfection (16), it is becoming increasingly difficult to combat the spread of this disease, and the global health burden of tuberculosis is extremely heavy. The reasons for the persistence of the tubercle bacillus include not only its ability to enter into a state of dormancy in its host for decades, evading the immune system by forming structures called granulomas (17), but also its unique and complex cell wall composed of specific lipids (8). These characteristics are thought to be good focus points for drug development. In granulomas, during the nonreplicative stage, the bacteria have been found to accumulate lipids in the form of intracellular lipid inclusion bodies (LIBs) (13). These lipids are composed mainly of triacylglycerols (TAG) (9, 13) and may originate from the lipolysis of host lipids and/or fatty acid uptake. In fact, M. tuberculosis in the granuloma center can even accumulate lipids originating from the degradation of immune cells (20). In addition, it has been reported that M. tuberculosis internalized by foamy macrophages accumulated LIBs when it joined cell lipid droplets composed of neutral lipids (32). Lipid storage may provide the bacillus with energy via the β-oxidation pathway followed by the glyoxylate cycle, during the chronic phase and the reactivation step (3, 17). These lipids may also supply precursors for the synthesis of bacterial cell membrane lipids, which play a key role in the pathogenicity of M. tuberculosis (4, 23). To investigate the molecular basis of the virulence and pathogenicity of M. tuberculosis, it was therefore proposed to study the lipid metabolism and cell wall remodeling processes in this bacterium.The enzymes involved in the lipid degradation processes induced by this bacterium have attracted considerable attention during the last few years. Based on the complete M. tuberculosis H37Rv genome sequence (6), several open reading frames (ORFs) encoding proteins potentially involved in the lipid metabolism of this strain have been identified, among which are the two lipases from M. tuberculosis that have been purified and characterized so far. Deb et al. identified an enzyme, Rv3097c (LipY), belonging to the hormone-sensitive lipase family, which is able to hydrolyze long-chain TAG (10). A study of LIB mobilization in a lipY-deficient mutant has shown that LipY was involved in TAG hydrolysis under nutriment-deprived conditions (10). LipY may therefore be involved in the degradation of TAG stored during the dormant stage and the subsequent reactivation of the pathogen. In addition, electron microscopy immunolabeling studies of LipY clearly showed that the enzyme had a cell surface localization, thus in direct contact with the host immune system (28). The last identified lipase to date is a monoacylglycerol lipase annotated Rv0183 (7). Like LipY, Rv0183 is located in the cell wall, but its exact physiological function has not yet been elucidated. One hypothesis could be that, like some mammalian cells (e.g., adipocytes), M. tuberculosis expresses several lipolytic enzymes sequentially involved in the lipolysis of TAG (37). The Rv0183 enzyme is conserved in M. bovis (Mb0189) and M. leprae (ML2603), as well as in M. smegmatis (MSMEG_0220), a nonpathogenic mycobacterium which provides a useful model organism and a surrogate host for molecular analysis of M. tuberculosis (19). In order to decipher the cellular role of Rv0183 in M. tuberculosis H37Rv and its contribution to the lipid metabolism of this bacterium, biochemical studies were performed on the homologue MSMEG_0220. For this purpose, the MSMEG_0220 gene from M. smegmatis, encoding a protein showing 68% amino acid sequence identity with Rv0183, was cloned, and the recombinant MSMEG_0220 enzyme (rMSMEG_0220) was produced in Escherichia coli, purified, and biochemically characterized. An M. smegmatis mutant with an MSMEG_0220 disrupted gene was produced to investigate the physiological role of MSMEG_0220.     

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.     

The Influence of Variable Rainfall Frequency on Germination and Early Growth of Shade-Tolerant Dipterocarp Seedlings in Borneo

Climate change induced alterations to rainfall patterns have the potential to affect the regeneration dynamics of plant species, especially in historically everwet tropical rainforest. Differential species response to infrequent rainfall may influence seed germination and seedling establishment in turn affecting species distributions. We tested the role of watering frequency intervals (from daily to six-day watering) on the germination and the early growth of Dipterocarpaceae seedlings in Borneo. We used seeds that ranged in size from 500 to 20,000 mg in order to test the role of seed mass in mediating the effects of infrequent watering. With frequent rainfall, germination and seedling development traits bore no relationship to seed mass, but all metrics of seedling growth increased with increasing seed mass. Cumulative germination declined by 39.4% on average for all species when plants were watered at six-day intervals, and days to germination increased by 76.5% on average for all species from daily to six-day intervals. Final height and biomass declined on average in the six-day interval by 16% and 30%, respectively, but the percentage decrease in final size was greater for large-seeded species. Rooting depth per leaf area also significantly declined with seed mass indicating large-seeded species allocate relatively more biomass for leaf production. This difference in allocation provided an establishment advantage to large-seeded species when water was non-limiting but inhibited their growth under infrequent rainfall. The observed reduction in the growth of large-seeded species under infrequent rainfall would likely restrict their establishment in drier microsites associated with coarse sandy soils and ridge tops. In total, these species differences in germination and initial seedling growth indicates a possible niche axis that may help explain both current species distributions and future responses to climate change.     

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.