The late steps of plant nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control system that identifies and degrades mRNAs containing premature termination codons (PTCs). If translation terminates at a PTC, the UPF1 NMD factor binds the terminating ribosome and recruits UPF2 and UPF3 to form a functional NMD complex, which triggers the rapid decay of the PTC-containing transcript. Although NMD deficiency is seedling lethal in plants, the mechanism of plant NMD remains poorly understood. To understand how the formation of the NMD complex leads to transcript decay we functionally mapped the UPF1 and SMG7 plant NMD factors, the putative key players of NMD target degradation. Our data indicate that the cysteine-histidine-rich (CH) and helicase domains of UPF1 are only essential for the early steps of NMD, whereas the heavily phosphorylated N- and C-terminal regions play a redundant but essential role in the target transcript degradation steps of NMD. We also show that both the N- and the C-terminal regions of SMG7 are essential for NMD. The N terminus contains a phosphoserine-binding domain that is required for the early steps of NMD, whereas the C terminus is required to trigger the degradation of NMD target transcripts. Moreover, SMG7 is a P-body component that can also remobilize UPF1 from the cytoplasm into processing bodies (P bodies). We propose that the N- and C-terminal phosphorylated regions of UPF1 recruit SMG7 to the functional NMD complex, and then SMG7 transports the PTC-containing transcripts into P bodies for degradation.

Biochemical characterization of the major N-acetylmuramidase from Lactobacillus buchneri.

Bacterial cell wall hydrolases are essential for peptidoglycan remodelling in regard to bacterial cell growth and division. In this study, peptidoglycan hydrolases (PGHs) of different Lactobacillus buchneri strains were investigated. First, the genome sequence of L. buchneri CD034 and L. buchneri NRRL B-30929 was analysed in silico for the presence of PGHs. Of 23 putative PGHs with different predicted hydrolytic specificities, the glycosyl hydrolase family 25 domain-containing homologues LbGH25B and LbGH25N from L. buchneri CD034 and NRRL B-30929, respectively, were selected and characterized in detail. Zymogram analysis confirmed hydrolysing activity on bacterial cell walls for both enzymes. Subsequent reversed-phase HPLC and MALDI-TOF MS analysis of the peptidoglycan breakdown products from L. buchneri strains CD034 and NRRL B-30929, and from Lactobacillus rhamnosus GG, which served as a reference, revealed that LbGH25B and LbGH25N have N-acetylmuramidase activity. Both enzymes were identified as cell wall-associated proteins by means of immunofluorescence microscopy and cellular fractionation, as well as by the ability of purified recombinant LbGH25B and LbGH25N to bind to L. buchneri cell walls in vitro. Moreover, similar secondary structures mainly composed of ß-sheets and nearly identical thermal stabilities with Tm values around 49 °C were found for the two N-acetylmuramidases by far-UV circular dichroism spectroscopy. The functional and structural data obtained are discussed and compared to related PGHs. In this study, a major N-acetylmuramidase from L. buchneri was characterized in detail for the first time.

In search of expression bottlenecks in recombinant CHO cell lines--a case study.

The efficient production of recombinant proteins such as antibodies typically involves the screening of an extravagant number of clones in order to finally select a stable and high-producing cell line. Thereby, the underlying principles of a powerful protein machinery, but also potential expression limitations, often remain poorly understood. To shed more light on this topic, we applied several different techniques to investigate a previously generated cell line (4B3-IgA), which expressed recombinant immunoglobulin A (IgA) with an unusually low specific productivity. Results were compared to the host cell line and to another recombinant CHO cell line (3D6-IgA) expressing another IgA that binds to an overlapping epitope. The low specific productivity of clone 4B3-IgA could not be explained by GCN or mRNA levels, but insufficiencies in protein maturation and/or secretion were determined. Despite the presence of free light chain polypeptides, they occasionally failed to associate with their heavy chain partners. Consequently, heavy chains were misassembled and accumulated to form intracellular aggregates, so-called Russell bodies. These protein deposits evoked the expression of increased amounts of ER-resident chaperones to combat the induced stress. Despite bottlenecks in protein processing, the cells' quality checkpoints remained intact, and predominantly correctly processed IgA was exported into the culture medium. The results of our study demonstrated that recombinant protein expression was impaired by heavy chain aggregation despite the presence of a disposable light chain and revealed elevated chaperone formation in combination with limited antibody assembly. Our studies suggest that the primary amino acid sequence and consequently the resulting structure of an expressed protein need to be considered as a factor influencing a cell's productivity.

Root Hair Imaging Using Confocal Microscopy.

Plant genetics plays a key role in determining root hair initiation and development. A complex network of genetic interactions therefore closely monitors and influences root hair phenotype and morphology. The significance of these genes can be studied by employing, for instance, loss-of-function mutants, overexpression plant lines, and fluorescently labeled constructs. Confocal laser scanning microscopy is a great tool to visually observe and document these morphological features. This chapter elaborates the techniques involved in handling of microscopic setup to acquire images displaying root hair distribution along the fully elongated zone of Arabidopsis thaliana roots. Additionally, we illustrate an approach to visualize early fate determination of epidermal cells in the root apical meristem, by describing a method for imaging YFP tagged transgenic plant lines.

S-layer coated emulsomes as potential nanocarriers.

The present study introduces a novel nanocarrier system comprising lipidic emulsomes and S-layer (fusion) proteins as functionalizing tools coating the surface. Emulsomes composed of a solid tripalmitin core and a phospholipid shell are created reproducibly with an average diameter of approximately 300 nm using temperature-controlled extrusion steps. Both wildtype (wt) and recombinant (r) S-layer protein SbsB of Geobacillus stearothermophilus PV72/p2 are capable of forming coherent crystalline envelope structures with oblique (p1) lattice symmetry, as evidenced by transmission electron microscopy. Upon coating with wtSbsB, positive charge of emulsomes shifts to a highly negative zeta potential, whereas those coated with rSbsB become charge neutral. This observation is attributed to the presence of a negatively charged glycan, the secondary cell wall polymer (SCWP), which is associated only with wtSbsB. The present study shows for the first time the ability of recombinant and wildtype S-layer proteins to cover the entire surface of emulsomes with its characteristic crystalline lattice. Furthermore, in vitro cell culture studies reveal that S-layer coated emulsomes can be uptaken by human liver carcinoma cells (HepG2) without showing any significant cytotoxicity over a wide range of concentrations. The utilization of S-layer fusion proteins equipped in a nanopatterned fashion by identical or diverse functions may lead to further development of emulsomes in nanomedicine, especially for drug delivery and targeting.

Identification of thylakoid membrane thermal transitions in Synechocystis sp. PCC6803 photosynthetic mutants.

We used differential scanning calorimetry (DSC) as a technique capable of identifying photosynthetic complexes on the basis of their calorimetric transitions. Annotation of thermal transitions was carried out with thylakoid membranes isolated from various photosynthetic mutants of Synechocystis sp. PCC6803. The thylakoid membranes exhibited seven major DSC bands between 40 and 85°C. The heat sorption curves were analyzed both by mathematical deconvolution of the overall endotherms and by a subsequent annealing procedure. The successive annealing procedure proved to be more reliable technique than mathematical deconvolution in assigning thermal transitions. The main DSC band, around 47°C, resulting from the high enthalpy change that corresponds to non-interacting complex of PSII, was assigned using the PSI-less/apcE(-) mutant cells. Another band around 68-70°C relates to the denaturation of PSII surrounded by other proteins of the photosynthetic complexes in wild type and PSI-less/apcE(-) cells. A further major transition found at 82-84°C corresponds to the PSI core complex of wild type and PSII-deficient BE cells. Other transition bands between 50-67 and 65-75°C are believed to relate to ATP synthase and cytochrome b(6)f, respectively. These thermal transitions were obtained with thylakoids isolated from PSI(-)/PSII(-) mutant cells. Some minor bands determined at 59 and 83-84°C correspond to an unknown complex and NADH dehydrogenase, respectively. These annotations were done by PSI-less/apcE(-) and PSI(-)/PSII(-) mutants.

A Novel, Broad-Acting Peptide Inhibitor of Double-Stranded DNA Virus Gene Expression and Replication.

Viral infections are a global disease burden with only a limited number of antiviral agents available. Due to newly emerging viral pathogens and increasing occurrence of drug resistance, there is a continuous need for additional therapeutic options, preferably with extended target range. In the present study, we describe a novel antiviral peptide with broad activity against several double-stranded DNA viruses. The 22-mer peptide TAT-I24 potently neutralized viruses such as herpes simplex viruses, adenovirus type 5, cytomegalovirus, vaccinia virus, and simian virus 40 in cell culture models, while being less active against RNA viruses. The peptide TAT-I24 therefore represents a novel and promising drug candidate for use against double-stranded DNA viruses.

Oxygen and temperature-dependent structural and redox changes in a novel cytochrome c(4) from the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina.

A novel cytochrome c(4), the first of this type in purple phototrophic bacteria has been discovered in Thiocapsa roseopersicina. The fact that cytochrome c(4) has been found in an anaerobic organism puts in question the up hereto suggested role of cytochromes c(4) in the aerobic respiratory metabolism. The structure of cytochrome c(4) was studied under both aerobic and anaerobic conditions, using differential scanning calorimetry and a combination of redox potentiostatic measurements with CD and UV-Vis absorption techniques. Cytochrome c(4) maintained its functional capability at high temperature (60 degrees C) if it was kept under anaerobic conditions. With increasing temperature under aerobic conditions, however, there are dramatic conformational changes in the protein and coordination changes on the iron side. Presumably oxygen binds to the iron at the position left vacant by the methionine and facilitates conformational changes with low reversibility.

Irreversible photoinhibition of photosystem II is caused by exposure of Synechocystis cells to strong light for a prolonged period.

Irreversible photoinhibition of photosystem II (PSII) occurred when Synechocystis sp. PCC 6803 cells were exposed to very strong light for a prolonged period. When wild-type cells were illuminated at 20 degrees C for 2 h with light at an intensity of 2,500 micromol photons m(-2) s(-1), the oxygen-evolving activity of PSII was almost entirely and irreversibly lost, whereas the photochemical reaction center in PSII was inactivated only reversibly. The extent of irreversible photoinhibition was enhanced at lower temperatures and by the genetically engineered rigidification of membrane lipids. Western and Northern blotting demonstrated that, after cells had undergone irreversible photoinhibition, the precursor to D1 protein in PSII was synthesized but not processed properly. These observations may suggest that exposure of Synechocystis cells to strong light results in the irreversible photoinhibition of the oxygen-evolving activity of PSII via impairment of the processing of pre-D1 and that this effect of strong light is enhanced by the rigidification of membrane lipids.

Streptococcal antigen I/II binds to extracellular proteins through intermolecular beta-sheets.

One of the functions associated with the oral streptococcal surface protein I/II is to bind to human extracellular matrix molecules or blood components, which could act as opportunistic ligands in pathological circumstances. In order to understand the relative specificity of the binding repertoire of this bacterial adhesin, we examined by infrared measurements the mode of binding of the protein I/II from Streptococcus mutans OMZ175 (I/IIf) to fibronectin and fibrinogen. This approach revealed the beta-structure forming capacity of I/IIf upon interaction with both proteins. The forming of intermolecular beta-structures may provide a non-selective way of interaction between I/IIf and its possible targets.

Plant peptides govern terminal differentiation of bacteria in symbiosis.

Legume plants host nitrogen-fixing endosymbiotic Rhizobium bacteria in root nodules. In Medicago truncatula, the bacteria undergo an irreversible (terminal) differentiation mediated by hitherto unidentified plant factors. We demonstrated that these factors are nodule-specific cysteine-rich (NCR) peptides that are targeted to the bacteria and enter the bacterial membrane and cytosol. Obstruction of NCR transport in the dnf1-1 signal peptidase mutant correlated with the absence of terminal bacterial differentiation. On the contrary, ectopic expression of NCRs in legumes devoid of NCRs or challenge of cultured rhizobia with peptides provoked symptoms of terminal differentiation. Because NCRs resemble antimicrobial peptides, our findings reveal a previously unknown innovation of the host plant, which adopts effectors of the innate immune system for symbiosis to manipulate the cell fate of endosymbiotic bacteria.

Low-temperature-induced accumulation of xanthophylls and its structural consequences in the photosynthetic membranes of the cyanobacterium Cylindrospermopsis raciborskii: an FTIR spectroscopic study.

The effects of the growth temperature on the lipids and carotenoids of a filamentous cyanobacterium, Cylindrospermopsis raciborskii, were studied., The relative amounts of polyunsaturated glycerolipids and myxoxanthophylls in the thylakoid membranes increased markedly when this cyanobacterium was grown at 25 degrees C instead of 35 degrees C. Fourier transform infrared spectroscopy was used to analyze the low-temperature-induced structural alterations in the thylakoid membranes. Despite the higher amount of unsaturated lipids there, conventional analysis of the v(sym)CH(2) band (characteristic of the lipid disorder) revealed more tightly arranged fatty-acyl chains for the thylakoids in the cells grown at 25 degrees C as compared with those grown at 35 degrees C. This apparent controversy was resolved by a two-component analysis of the v(sym)CH(2) band, which demonstrated very rigid, myxoxanthophyll-related lipids in the thylakoid membranes. When this rigid component was excluded from the analysis of the thermotropic responses of the v(sym)CH(2) bands, the expected higher fatty-acyl disorder was observed for the thylakoids prepared from cells grown at 25 degrees C as compared with those grown at 35 degrees C. Both the carotenoid composition and this rigid component in the thylakoid membranes were only growth temperature-dependent; the intensity of the illuminating light during cultivation had no apparent effect on these parameters. We propose that, besides their well-known protective functions, the polar carotenoids in particular may have structural effects on the thylakoid membranes. These effects should be exerted locally--by forming protective patches, in-membrane barriers of low dynamics--to prevent the access of reactive radicals generated in either enzymatic or photosynthetic processes to sensitive spots of the membranes.