Glycosylation and functionality of recombinant β-glucocerebrosidase from various production systems

The glycosylation of recombinant β-glucocerebrosidase, and in particular the exposure of mannose residues, has been shown to be a key factor in the success of ERT (enzyme replacement therapy) for the treatment of GD (Gaucher disease). Macrophages, the target cells in GD, internalize β-glucocerebrosidase through MRs (mannose receptors). Three enzymes are commercially available for the treatment of GD by ERT. Taliglucerase alfa, imiglucerase and velaglucerase alfa are each produced in different cell systems and undergo various post-translational or post-production glycosylation modifications to expose their mannose residues. This is the first study in which the glycosylation profiles of the three enzymes are compared, using the same methodology and the effect on functionality and cellular uptake is evaluated. While the major differences in glycosylation profiles reside in the variation of terminal residues and mannose chain length, the enzymatic activity and stability are not affected by these differences. Furthermore, the cellular uptake and in-cell stability in rat and human macrophages are similar. Finally, in vivo studies to evaluate the uptake into target organs also show similar results for all three enzymes. These results indicate that the variations of glycosylation between the three regulatory-approved β-glucocerebrosidase enzymes have no effect on their function or distribution.     

Solubilization and interaction of α-tocopherol in water-aerosol OT-isooctane systems

Solubilization and interaction of α-tocopherol into bis(2-ethylhexyl)sulphosuc cinate sodium salt microemulsion systems have been studied by temperature dependent phase transition, viscosity and nuclear magnetic resonance studies. Tocopherol being an amphiphilic molecule dissolves into the interfacial surfactant monolayer of the microemul sion droplets. The dissolution leads to an enhancement of the rigidity of the surfactant monolayer as studied by the increase in mixing and phase transition temperatures of the microemulsion droplets. Solubilization of tocopherol into microemulsion droplets causes an increase in the effective size of the droplet and as a consequence, the inter-droplet interactions are also increased. The water binding capacity of the surfactant (bis(2-ethylhexyl)sulphosuccinate sodium salt) is reduced due to solubilization of tocopherol as is evidenced from the downfield shifts of water proton magnetic resonances. In the presence of the dissolved electrolytes into the aqueous core, tocopherol is squeezed out of the microemulsion droplets increasing the membrane fluidity and permeability.     

Isolation and characterization of phage–host systems from the Baltic Sea ice

In search for sea ice bacteria and their phages from the Baltic Sea ice, two ice samples were collected from land-fast ice in a south-west Finland coastal site in February and March 2011. Bacteria were isolated from the melted sea ice samples and phages were screened from the same samples for 43 purified isolates. Plaque-producing phages were found for 15 bacterial isolates at 3 °C. Ten phage isolates were successfully plaque purified and eight of them were chosen for particle purification to analyze their morphology and structural proteins. Phage 1/32 infecting an isolate affiliated to phylum Bacteroidetes (Flavobacterium sp.) is a siphovirus and six phages infecting isolates affiliated to γ-Proteobacteria (Shewanella sp.) hosts were myoviruses. Cross titrations between the hosts showed that all studied phages are host specific. Phage solutions, host growth and phage infection were tested in different temperatures revealing phage temperature tolerance up to 45 °C, whereas phage infection was in most of the cases retarded above 15 °C. This study is the first to report isolation and cultivation of ice bacteria and cold-active phages from the Baltic Sea ice.     

Activity and stability of Escherichia coli β-galactosidase in cosolvent systems

The kinetic parameters of E.coli -galactosidase were not altered by the addition of 2-propanol or ethyl acetate (1.6% v/v). While ethylene glycol (1.6% v/v) doubled the values of both KM (0.29 mM) and kcat (1393 s–), tetraethyleneglycol-dimethylether (Tetraglyme,1.6% v/v) preserved KM, but decreased kcat. At 50°C all the cosolvents dramatically shortened the enzymatic half life, and so did Tetraglyme and 2-propanol at 28°C. At 28°C, both ethyl acetate and ethylene glycol stabilised the enzyme 9- and 6-fold respectively. This fact, together with the activation effect of ethylene glycol may lead to practical applications. © Rapid Science Ltd. 1998     

Partitioning of α-lactalbumin and β-lactoglobulin in aqueous two-phase systems of polyvinylpyrrolidone and potassium phosphate

In the present study, the partitioning of α-lactalbumin, β-lactoglobulin, and cheese whey proteins in aqueous two-phase system of polyvinylpyrrolidone-potassium phosphate is investigated. The partitioning of proteins in this system depends on the polymer and salt weight percents in feed, temperature, and pH. The orthogonal central composite design is used to study the effects of different parameters on partitioning of α-lactalbumin and β-lactoglobulin. A second order model is proposed to determine the impact of these parameters. The results of the model show that the weight percent of the salt in feed has a large effect on the protein partitioning. The weight percent of polyvinylpyrrolidone in the feed increases the partitioning coefficients. By increasing the temperature, the viscosity of polyvinylpyrrolidone is reduced and the protein can easily be transferred from one phase to the other phase. The pH of the aqueous two phase system can alter the protein partitioning coefficient through the variation of the protein net charge.     

Thermal acclimation,heat shock and photoperiod: Do these factors interplay in the adaptive responses of crab neuromuscular systems to temperature?

Evidence is reviewed demonstrating that the adaptive responses to temperature made by the walking leg neuromuscular system of crabs (Carcinus maenas) are in response to local temperature and not in response to hierarchical influences by the CNS and hormonal systems.     

Proliferation and degeneration of circulating CFU-GM in 15 adult normal subjects. Can any ontogenetic relationship be deduced from these data?

We studied, in 15 normal adults, the "in vitro" proliferation and differentiation of circulating CFU-GM, in order to assess their implication in the processes that regulate the dynamic equilibrium of granulopoiesis, analogous to bone marrow CFU-GM, and to deduce by their growth behaviour the ontogenetic relationship between CFU-GM subpopulations in the circulating and bone marrow compartments respectively. We found that "in vitro" proliferation of circulating CFU-GM predominates over their degeneration. We believe circulating CFU-GM and bone marrow CFU-GM are not implicated in granulopoiesis regulation in the same manner, and that circulating CFU-GM are more immature than bone marrow CFU-GM when taking proliferation and GM-CSF response into account. One cannot ignore the hypothesis that cells that "in vivo" are quiescent, are recruited "in vitro" with GM-CSF. Finally we would like to draw attention to the parallelism between CFU-GM classification in types 1 and 2 using monoclonal antibodies to track surface antigens, and our classification obtained by using a new mathematical model that takes the "birth" and "dead" of cellular aggregates into account.     

Characterization and comparative analysis of toxin–antitoxin systems in Acetobacter pasteurianus

Journal of Industrial Microbiology & Biotechnology - Bacterial toxin–antitoxin (TA) systems play important roles in diverse cellular regulatory processes. Here, we characterize three...     

Glucosylceramides of oat root plasma membranes — physicochemical behaviour in natural and in model systems

Glucosylceramides from oat root plasma membranes have been characterized using HPLC-particle beam-mass spectrometry, differential scanning calorimetry, low angle X-ray diffraction and surface balance technique. 24:1-OH was dominating fatty acid (90%) together with 24:0-OH and 22:1-OH. The sphingosine base was sphingadienine isomers and the monosaccharide α and β glucose. Differential scanning calorimetry of an aqueous dispersion of glucosylceramide revealed during heating an endothermic gel-liquid crystalline transition with double peaks at 47° and 51°C, the lowest known for naturally occurring glucosylceramides. A cooling scan after the endothermic gel-liquid transition showed one exotherm at 15°C and if this was followed by another heating scan a large exotherm appeared with a peak at 18°C. During the second heating the matrix was hydrated and the exotherm at 18°C reflects then the transition between the supercooled metastable gel phase and the corresponding hydrated form. The calorimetric data indicate a lamellar phase which during the cooling scan appeared as an supercooled liquid crystalline phase. Low angle X-ray diffraction confirmed these calorimetric data. The surface pressure-area-curves of pure oat glucosylceramides were more expanded than those of bovine origin. Mixtures of oat glucosylceramides and phosphatidylcholine species similar to those present in oat root plasma membranes showed molecular miscibility but no interaction.     

Type II and VI collagen in nasal and articular cartilage and the effect of IL-1α on the distribution of these collagens

The distribution of type II and VI collagen was immunocytochemically investigated in bovine articular and nasal cartilage. Cartilage explants were used either fresh or cultured for up to 4 weeks with or without interleukin 1α (IL-1α). Sections of the explants were incubated with antibodies for both types of collagen. Microscopic analyses revealed that type II collagen was preferentially localized in the interchondron matrix whereas type VI collagen was primarily found in the direct vicinity of the chondrocytes. Treatment of the sections with hyaluronidase greatly enhanced the signal for both types of collagen. Also in sections of explants cultured with IL-1α a higher level of labeling of the collagens was found. This was apparent without any pre-treatment with hyaluronidase. Under the influence of IL-1α the area positive for type VI collagen that surrounded the chondrocytes broadened. Although the two collagens in both types of cartilage were distributed similarly, a remarkable difference was the higher degree of staining of type VI collagen in articular cartilage. Concomitantly we noted that digestion of this type of cartilage hardly occurred in the presence of IL-1α whereas nasal cartilage was almost completely degraded within 18 days of culture. Since type VI collagen is known to be relatively resistant to proteolysis we speculate that the higher level of type VI collagen in articular cartilage is important in protecting cartilage from digestion.     

S-Adenosyl-l-methionine–cycloartenol methyltransferase activity in cell-free systems from Trebouxia sp. and Scenedesmus obliquus

1. Homogenates prepared from Trebouxia sp. 213/3 and Scenedesmus obliquus exhibited S-adenosyl-l-methionine-cycloartenol methyltransferase activity. 2. The products of the reaction, with cycloartenol as the substrate, were 24-methylenecycloartanol and cyclolaudenol. 3. Optimal enzyme activity was found in homogenates prepared at pH7.6 and the transmethylase was distributed between the supernatant and microsomal fractions of the Trebouxia homogenate. 4. The relevance of these results is discussed in relation to C(28) and C(29) sterol production in the algae.     

Bacteriophytochromes – from informative model systems of phytochrome function to powerful tools in cell biology

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Importance of adaptation and genotype × environment interactions in tropical beef breeding systems

This paper examines the relative importance of productive and adaptive traits in beef breeding systems based on Bos taurus and tropically adapted breeds across temperate and (sub)tropical environments. In the (sub)tropics, differences that exist between breeds in temperate environments are masked by the effects of environmental stressors. Hence in tropical environments, breeds are best categorised into breed types to compare their performance across environments. Because of the presence of environmental stressors, there are more sources of genetic variation in tropical breeding programmes. It is therefore necessary to examine the genetic basis of productive and adaptive traits for breeding programmes in those environments. This paper reviews the heritabilities and genetic relationships between economically important productive and adaptive traits relevant to (sub)tropical breeding programmes. It is concluded that it is possible to simultaneously genetically improve productive and adaptive traits in tropically adapted breeds of beef cattle grazed in tropical environments without serious detrimental consequences for either adaptation or production. However, breed-specific parameters are required for genetic evaluations. The paper also reviews the magnitude of genotype × environment (G × E) interactions impacting on production and adaptation of cattle, where 'genotype' is defined as breed (within a crossbreeding system), sire within breed (in a within-breed selection programme) or associations between economically important traits and single nucleotide polymorphisms (SNPs - within a marker-assisted selection programme). It is concluded that re-ranking of breeds across environments is best managed by the use of the breed type(s) best suited to the particular production environment. Re-ranking of sires across environments is apparent in poorly adapted breed types across extreme tropical and temperate environments or where breeding animals are selected in a temperate environment for use in the (sub)tropics. However, G × E interactions are unlikely to be of major importance in tropically adapted beef cattle grazed in either temperate or (sub)tropical environments, although sex × environment interactions may provide new opportunities for differentially selecting to simultaneously improve steer performance in benign environments and female performance in harsher environments. Early evidence suggests that re-ranking of SNPs occurs across temperate and tropical environments, although their magnitude is still to be confirmed in well-designed experiments. The major limitation to genetic improvement of beef cattle over the next decade is likely to be a deficiency of large numbers of accurately recorded phenotypes for most productive and adaptive traits and, in particular, for difficult-to-measure adaptive traits such as resistance to disease and environmental stressors.     

Variability and specialization of plant–pollinator systems in a northern maritime grassland

To detect plant specialization to pollinator insects and to further examine the pollination syndrome hypotheses, flowering angiosperms and insect visitors were recorded in a northern maritime grassland community. Associations between plant species and insect groups were analyzed using binomial tests based on census data obtained from two sites over 3years. Preference to an insect group by a plant species was expressed as a significant deviation of the actual proportion (i.e. the proportion of the number of flower visits by the insect group to the plant species in the total number of visits to the plant species) of the expected proportion (i.e. the proportion of the number of visits by the insect group in the total visits during its flowering period). Preference to a plant species by an insect group was similarly expressed using the number of flowers of the plant species visited by the insect group. Most significant preferences varied temporally or spatially. Variability in the preferences is suggested to have resulted from temporal and spatial variations in the abundance and species composition of both flowers and insects. However, in species showing variable preferences, significant specialization in the pollinator insect group (i.e. relatively constant, mutual preferences) were demonstrated for seven and five plant species at the two study sites, respectively. Most specializations were found in associations with bumblebees. Bumblebee specialists were significantly well represented in the flower shape types gullet and flag and the flower color types violet, which supports the pollination syndrome theory.     

Sustainability criteria and indicators of bioenergy systems from steering,research and Finnish bioenergy business operators’ perspectives

This paper addresses the interface of steering, research, and business operators’ perspectives to bioenergy sustainability. Although bioenergy business operators are essential for sustainable development of bioenergy systems through implementation of sustainability criteria, their perspective to sustainability is rarely studied. We systematically studied the relevant sustainability criteria and indicators from the three perspectives in different stages of a general bioenergy life cycle and in different sustainability dimensions, and evaluated bioenergy operators’ sustainability principles, criteria and indicators simultaneously with respect to the steering and research data and a business sustainability maturity framework. We collected data from literature and a workshop for Finnish bioenergy experts. The results show a similar division of steering and operators’ sustainability criteria and indicators to life cycle stages and sustainability dimensions with a slight emphasis on business economic sustainability from operators’ perspective. The acceptability principle could provide bioenergy operators a meaningful way of identifying the role of sustainability criteria and indicators from steering and research sources in advancing their business sustainability maturity.     

Meat and fat quality of Krškopolje pigs reared in conventional and organic production systems

Data on production traits of the only Slovenian autochthonous pig breed, the Krškopolje pig, is very scarce. Krškopolje pigs are reared in conventional and organic production systems, which were compared in the present study. After weaning, 24 barrows were assigned within litter to either conventional (CON) or organic (ECO) rearing system. Group CON (n=12) was housed indoors in two pens (7.5 m²) with partly slatted floor. Group ECO (n=12) was held in a sty with sheltered area (concrete floor, bedded with straw, 16 m²) and outdoor paddock area (100 m²). The trial started when pigs had 68±8 kg BW and 157±6 days of age. Two diets were formulated with equivalent ingredients and composition. For ECO diet the ingredients used were ecological. Group ECO received a diet with 12.4 MJ metabolisable energy (ME) per kilogram and 12.9% CP and group CON a diet with 12.7 MJ ME/kg and 13.6% CP. Feed distribution was limited to 3.5 kg per pig daily. In line with the rules for organic production, ECO pigs were additionally given alfalfa hay ad libitum. After 73 days on trial, the pigs were slaughtered and carcass, meat and fat quality was evaluated. Meat quality traits (pH, colour, water holding capacity), fatty acid composition, lipid and protein oxidation, collagen content and solubility were analysed in longissimus lumborum (LL) muscle. Fatty acid composition, lipid oxidation, and vitamins A and E concentrations were determined in backfat. There were no significant differences in growth rate and carcass traits between ECO and CON pigs, however, ECO pigs tended (P<0.10) to have higher daily gain and lower dressing percentage, higher (P<0.001) pH 45 min and lower (P<0.01) pH 24 h postmortem, affecting (P<0.10) also water holding capacity and objective colour parameters (P<0.05) of LL muscle. There were no differences in intramuscular fat (IMF) content of LL muscle, however, IMF of ECO pigs had lower (P<0.05) proportion of saturated and higher (P<0.01) proportion of monounsaturated fatty acids accompanied by higher (P<0.001) values of thiobarbituric reactive substances (TBARS). In backfat, ECO pigs showed lower (P<0.05) vitamin E content, higher (P<0.001) TBARS, higher (P<0.01) degree of unsaturation (percentage of polyunsaturated fatty acids), and also higher (P<0.05) vitamin A concentration than CON pigs, which can be related to alfalfa hay supplementation of ECO pigs. In brief, organic rearing of Krškopolje pigs did not affect performances but had an effect on meat and fat quality.     

Involvement of GABAergic and glutamatergic systems in the anticonvulsant activity of 3-alkynyl selenophene in 21?day-old rats

In this study, we investigated the role of GABAergic and glutamatergic systems in the anticonvulsant action of 3-alkynyl selenophene (3-ASP) in a pilocarpine (PC) model of seizures. To this purpose, 21 day-old rats were administered with an anticonvulsant dose of 3-ASP (50 mg/kg, per oral, p.o.), and [(3)H]γ-aminobutyric acid (GABA) and [(3)H]glutamate uptakes were carried out in slices of cerebral cortex and hippocampus. [(3)H]GABA uptake was decreased in cerebral cortex (64%) and hippocampus (58%) slices of 21 day-old rats treated with 3-ASP. In contrast, no alteration was observed in [(3)H]glutamate uptake in cerebral cortex and hippocampus slices of 21 day-old rats that received 3-ASP. Considering the drugs that increase synaptic GABA levels, by inhibiting its uptake or catabolism, are effective anticonvulsants, we further investigated the possible interaction between sub-effective doses of 3-ASP and GABA uptake or GABA transaminase (GABA-T) inhibitors in PC-induced seizures in 21 day-old rats. For this end, sub-effective doses of 3-ASP (10 mg/kg, p.o.) and DL-2,4-diamino-n-butyric acid hydrochloride (DABA, an inhibitor of GABA uptake--2 mg/kg, intraperitoneally; i.p.) or aminooxyacetic acid hemihydrochloride (AOAA; a GABA-T inhibitor--10 mg/kg, i.p.) were co-administrated to 21 day-old rats before PC (400 mg/kg; i.p.) treatment, and the appearance of seizures was recorded. Results demonstrated that treatment with AOAA and 3-ASP or DABA and 3-ASP significantly abolished the number of convulsing animals induced by PC. The present study indicates that 3-ASP reduced [(3)H]GABA uptake, suggesting that its anticonvulsant action is related to an increase in inhibitory tonus.