Protein chemical characterization of Mucor pusillus aspartic proteinase. Amino acid sequence homology with the other aspartic proteinases, disulfide bond arrangement and site of carbohydrate attachment

The amino acid sequence of Mucor pusillus aspartic proteinase was determined by analysis of fragments obtained from cleavage of the enzyme by CNBr and limited tryptic digestion. The proteinase is a single polypeptide chain protein containing 361 amino acid residues, cross-linked by two disulfide bonds. A sugar moiety composed of two GlcNAc residues and four neutral sugar residues is asparagine-linked to the chain. The sequence of M. pusillus proteinase is highly homologous with the M. miehei proteinase (83% identity). The homology with other aspartic proteinases is low (22-24%) and indicates that the Mucor proteinases diverged at an early evolutionary phase. The most conservative regions of the molecule are those involved in catalysis and forming the binding cleft and the core region of the molecule.     

Community Seasonal Development Enables Late Defoliation Without Loss of Forage Quality in Semi-natural Grasslands

Agri-environmental measures often require postponing of grassland defoliation until summer months. We investigated how this affects agronomic characteristics, i.e., biomass production and forage quality, in species-rich grasslands in the White Carpathian Mountains, Czech Republic. Three distinct grasslands (vegetation alliances Bromion erecti, Cynosurion cristati, Violion caninae) were selected for biomass sampling in three dates: mid-May, early June and end of June. Proportions of individual species from total biomass, biomass production, crude fibre concentration, organic matter digestibility and community functional properties (forage value, leaf dry matter content, specific leaf area) were determined. Dry matter standing biomass at the end of June was highest in Bromion with 3.5 t/ha, followed by Violion with 2.7 t/ha and Cynosurion with 2.3 t/ha. A steep decline in forage quality (increase in crude fibre and decrease in organic matter digestibility) during accumulation of above-ground biomass was recorded in formerly unmanaged Bromion grassland but remarkably not in formerly grazed Cynosurion and Violion grasslands where early spring dominants were partly replaced by later developing species, Agrostis capillaris and Trifolium spp. Abundance-weighted community functional properties were consistent with results obtained from biomass chemical analyses, thus the ‘trait approach’ can be used as a suitable surrogate of costly and labor-intensive laboratory procedures. Finally, we assume that the high degree of community seasonal development in Cynosurion and Violion, indicated here by a new community seasonal development index and by development in community specific leaf area, was accountable for the stabilization of forage quality later in the vegetation season.     

Restoration of grazing management and its effect on vegetation in an upland grassland

Question: How are plant species and functional group composition, and potential sward height affected by implementation of different grazing regimes on previously abandoned semi-natural grassland? Location: The Jizerské mountains, northern Czech Republic. Methods: We established a randomized block experiment with the following treatments: unmanaged control (U), intensive (IG) and extensive (EG) continuous grazing, first cut followed by intensive (ICG) and first cut followed by extensive (ECG) continuous grazing for the rest of the growing season. The percentage cover of all vascular plant species was recorded in 40 permanent plots. Results: Total plant species richness increased in all managed treatments, whereas species number was reduced in U at the end of the experiment. Tall forbs (Aegopodium podagraria, Galium album, Anthriscus sylvestris, Cirsium arvense) as well as tall grasses (Elytrigia repens and Alopecurus pratensis) were more abundant in U. Species associated with both grazing treatments (IG, EG) were Dactylis glomerata, Festuca rubra agg. and Phleum pratense. Agrostis capillaris, Taraxacum spp., Trifolium repens, Ranunculus repens and Cirsium vulgare were promoted by ECG and ICG. Abundance of tall grasses and tall forbs reflected the intensity of management in the order U>EG, ECG>IG, ICG. Prostrate forbs, on the other hand, increased their cover with increasing intensity: ICG>IG>ECG>EG. Conclusions: Plant species composition of semi-natural grasslands is affected by the defoliation regime. Continuous grazing on abandoned grassland alters the sward structure towards a permanent pasture with short, light-sensitive grasses and prostrate forbs. To maintain or enhance plant species richness in semi-natural grasslands, understanding the effects of different grazing regimes on plant species composition is necessary.     

Effects of exogenous nitric oxide on photosynthesis

Nitric oxide (NO) is an important signalling molecule with diverse physiological functions in plants. In plant cell, it is synthesised in several metabolic ways either enzymatically or nonenzymatically. Due to its high reactivity, it could be also cytotoxic in dependence on concentration. Such effects could be also mediated by NO-derived compounds. However, the role of NO in photosynthetic apparatus arrangement and in photosynthetic performance is poorly understood as indicated by a number of studies in this field with often conflicting results. This review brings a short survey of the role of exogenous NO in photosynthesis under physiological and stressful conditions, particularly of its effect on parameters of chlorophyll fluorescence.     

A new approach to CAR T-cell gene engineering and cultivation using piggyBac transposon in the presence of IL-4, IL-7 and IL-21

Background aims

Clinical-grade chimeric antigenic receptor (CAR)19 T cells are routinely manufactured by lentiviral/retroviral (LV/RV) transduction of an anti-CD3/CD28 activated T cells, which are then propagated in a culture medium supplemented with interleukin (IL)-2. The use of LV/RVs for T-cell modification represents a manufacturing challenge due to the complexity of the transduction approach and the necessity of thorough quality control.

Cloning, biochemical properties, and distribution of mycobacterial haloalkane dehalogenases

Haloalkane dehalogenases are enzymes that catalyze the cleavage of the carbon-halogen bond by a hydrolytic mechanism. Genomes of Mycobacterium tuberculosis and M. bovis contain at least two open reading frames coding for the polypeptides showing a high sequence similarity with biochemically characterized haloalkane dehalogenases. We describe here the cloning of the haloalkane dehalogenase genes dmbA and dmbB from M. bovis 5033/66 and demonstrate the dehalogenase activity of their translation products. Both of these genes are widely distributed among species of the M. tuberculosis complex, including M. bovis, M. bovis BCG, M. africanum, M. caprae, M. microti, and M. pinnipedii, as shown by the PCR screening of 48 isolates from various hosts. DmbA and DmbB proteins were heterologously expressed in Escherichia coli and purified to homogeneity. The DmbB protein had to be expressed in a fusion with thioredoxin to obtain a soluble protein sample. The temperature optimum of DmbA and DmbB proteins determined with 1,2-dibromoethane is 45 degrees C. The melting temperature assessed by circular dichroism spectroscopy of DmbA is 47 degrees C and DmbB is 57 degrees C. The pH optimum of DmbA depends on composition of a buffer with maximal activity at 9.0. DmbB had a single pH optimum at pH 6.5. Mycobacteria are currently the only genus known to carry more than one haloalkane dehalogenase gene, although putative haloalkane dehalogenases can be inferred in more then 20 different bacterial species by comparative genomics. The evolution and distribution of haloalkane dehalogenases among mycobacteria is discussed.     

The evolution of symmetrical snapping in termite soldiers need not lead to reduced chemical defence

Termite soldiers use a multitude of defensive strategies, often combining mechanical weapons with defensive chemicals secreted from their frontal gland. In higher termites (Termitidae), both these weapons are situated in the head of the soldiers. Their simultaneous occurrence is supposed to be subjected to energetic and spatial constraints and the resulting trade‐off often leads to a full development of only one of the weapons and reduction of the other. Mandibular snapping represents an extreme and efficient anatomical adaptation, allowing the soldier to knock out arthropod enemies with a violent strike of elongated mandibles. The head of snapping soldiers harbours massive adductor muscles while the frontal gland is reported to be reduced and the chemical defence absent. Here we show that the symmetrical snapping soldiers of the Neotropical termite Cavitermes tuberosus possess a well‐developed frontal gland situated in the frontal projection on their head. The gland produces a blend of unbranched alkenes and alkadienes combined with three diterpene hydrocarbons, derived from a novel bicyclic hydrocarbon skeleton. We characterized the molecular structure of the most abundant of these diterpenes and proposed for it a trivial name, cavitene. We conclude that the evolution of the mandibular snapping has not necessarily been accompanied by the reduction or loss of chemical defensive adaptations and that the two defensive modes are not mutually incompatible. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ●● , ●●–●●.     

MOLE: a Voronoi diagram-based explorer of molecular channels, pores, and tunnels

We have developed an algorithm, "MOLE," for the rapid, fully automated location and characterization of molecular channels, tunnels, and pores. This algorithm has been made freely available on the Internet (http://mole.chemi.muni.cz/) and overcomes many of the shortcomings and limitations of the recently developed CAVER software. The core of our MOLE algorithm is a Dijkstra's path search algorithm, which is applied to a Voronoi mesh. Tests on a wide variety of biomolecular systems including gramicidine, acetylcholinesterase, cytochromes P450, potassium channels, DNA quadruplexes, ribozymes, and the large ribosomal subunit have demonstrated that the MOLE algorithm performs well. MOLE is thus a powerful tool for exploring large molecular channels, complex networks of channels, and molecular dynamics trajectories in which analysis of a large number of snapshots is required.     

A Solid Phase Vibrational Circular Dichroism Study of Polypeptide–Surfactant Interaction

We studied the interaction of poly‐l ‐lysine (PLL) and poly‐l ‐arginine (PLAG) with sodium dodecyl sulfate (SDS) surfactant and the interaction of poly‐l‐ glutamic acid (PLGA) and poly‐l ‐aspartic acid (PLAA) with tetradecyltrimethylammonium bromide (TTAB) surfactant using vibrational circular dichroism (VCD) spectroscopy in the region of C‐H stretching vibration and in the Amide I region both in solution and in mulls. A chirality transfer from polypeptides to achiral surfactants was observed in the C‐H stretching region, where measurements in solution were impossible. This observation was enabled by a special sample treatment technique using lyophilization and the preparation of mulls. This technique demonstrated itself as an interesting and beneficial tool for VCD measurements. In addition, we observed that SDS changed the secondary structure of PLL to the β‐sheet and of PLAG to the α‐helix. TTAB disrupted the PLGA and PLAA structure. These results were obtained in the mull but were confirmed by the VCD spectra measured in solution and by electronic circular dichroism. The chirality transfer from the polypeptides to SDS was caused by polypeptides ordered into a specific conformation during the interaction, while in the TTBA system it was induced primarily by the chirality of the amino acid residues. Chirality 27:965–972, 2015. © 2015 Wiley Periodicals, Inc.