Characterization of Solanum tuberosum Multicystatin and Its Structural comparison with Other Cystatins

Potato (Solanum tuberosum) multicystatin (PMC) is a crystalline Cys protease inhibitor present in the subphellogen layer of potato tubers. It consists of eight tandem domains of similar size and sequence. Our in vitro results showed that the pH/PO₄⁻-dependent oligomeric behavior of PMC was due to its multidomain nature and was not a characteristic of the individual domains. Using a single domain of PMC, which still maintains inhibitor activity, we identified a target protein of PMC, a putative Cys protease. In addition, our crystal structure of a representative repeating unit of PMC, PMC-2, showed structural similarity to both type I and type II cystatins. The N-terminal trunk, α-helix, and L2 region of PMC-2 were most similar to those of type I cystatins, while the conformation of L1 more closely resembled that of type II cystatins. The structure of PMC-2 was most similar to the intensely sweet protein monellin from Dioscorephyllum cumminisii (serendipity berry), despite a low level of sequence similarity. We present a model for the possible molecular organization of the eight inhibitory domains in crystalline PMC. The unique molecular properties of the oligomeric PMC crystal are discussed in relation to its potential function in regulating the activity of proteases in potato tubers.     

Crystal Structure of an Ammonia-Permeable Aquaporin

Aquaporins of the TIP subfamily (Tonoplast Intrinsic Proteins) have been suggested to facilitate permeation of water and ammonia across the vacuolar membrane of plants, allowing the vacuole to efficiently sequester ammonium ions and counteract cytosolic fluctuations of ammonia. Here, we report the structure determined at 1.18 Å resolution from twinned crystals of Arabidopsis thaliana aquaporin AtTIP2;1 and confirm water and ammonia permeability of the purified protein reconstituted in proteoliposomes as further substantiated by molecular dynamics simulations. The structure of AtTIP2;1 reveals an extended selectivity filter with the conserved arginine of the filter adopting a unique unpredicted position. The relatively wide pore and the polar nature of the selectivity filter clarify the ammonia permeability. By mutational studies, we show that the identified determinants in the extended selectivity filter region are sufficient to convert a strictly water-specific human aquaporin into an AtTIP2;1-like ammonia channel. A flexible histidine and a novel water-filled side pore are speculated to deprotonate ammonium ions, thereby possibly increasing permeation of ammonia. The molecular understanding of how aquaporins facilitate ammonia flux across membranes could potentially be used to modulate ammonia losses over the plasma membrane to the atmosphere, e.g., during photorespiration, and thereby to modify the nitrogen use efficiency of plants.     

Reduced use of antimicrobials after vaccination of pigs against porcine proliferative enteropathy in a Danish SPF herd

The present study explored whether the use of group medication with antibiotics in a Danish pig herd was reduced after vaccination of the pigs against proliferative enteropathy (PE) caused by Lawsonia intracellularis. 7900 pigs originating from a single commercial sow herd were vaccinated against L. intracellularis, whereas 7756 pigs were kept as non-vaccinated controls. The pigs were included batch-wise in the study with every second batch being vaccinated. In the vaccinated batches, the consumption of oxytetracykline to treat PE was reduced by 79%, with a significantly lower number of pigs being treated (P < 0.0001). Vaccination also resulted in a highly significant improvement of average daily weight gain (+ 46 g/day; P = 9.55 × 10^-31) and carcase weight (+ 1.25 kg; P = 4.54 × 10^-05) as well as a shortened fattening period (-8 days; P = 2.01 × 10^-45).     

Ovulation and Embryonic Developmental Rate Following hCG-stimulation in Sows

The hCG induced ovulation in sows was studied by use of ultrasonography, and an investigation of the development and diversity of the zygotes/embryos was performed at 24 h after ovulation. Crossbred sows (N=48) were weaned (day 0) and checked for heat twice daily from day 3 onwards. From day 4, the ovaries were transrectally scanned twice daily On day 4, the sows were given an injection of 750 iu hCG im and inseminated 27 ± 2 h (X ± SD) and 38 ± 1 h later. From 38 to 48 h after the hCG injection, the ovaries were scanned at 60 to 90 min intervals. At 24 h after ovulation the oviducts were surgically flushed in 18 sows. Out of the 48 sows, 34 showed heat at 12–36 h after the hCG-treatment and 14 showed heat before the hCG treatment. In the former group of sows, 20 (59%) ovulated within the interval of 38 to 48 h after the hCG treatment, and the follicular size immediately before ovulation was 7.8 ± 0.6 mm. Among the sows which showed heat before hCG treatment only 7 (50%) ovulated within the above interval and the preovulatory follicle size was larger (8.3 ± 0.5, p<0.05) than in the former group of sows, which showed heat after the hCG treatment. The flushing of 18 sows yielded a total of 243 ova, 70 (29 %) 1-cell stages, 160 (66 %) 2-cell stages and 13 (5%) 4-cell stages. A pronounced difference in the degree of variation in embryonic development was seen between sows: 4 animals yielded 1- to 4-cell stages, one exclusively 2-cell stage. In conclusion, the control of ovulation in sows by hCG treatment will affect the follicular growth and the exact timing of ovulation can not always be relied on. It is strongly recommended to use ultrasonography to monitor the time of ovulation if this parameter is important. Ova recovered at 24±1 h after the median time of ovulation revealed a pronounced diversity (1- to 4- cell stage) within sows. No obvious relation with this embryonic diversity and the follicular size at ovulation was seen in these data.     

Steady-state and transient membrane potentials in human red cells determined by protonophore-mediated pH changes

Summary Protonophores have been used frequently to determine changes in membrane potential in suspensions of red cells, since such changes are reflected by changes in extracellular pH, due to proton and consequently protonophore reequilibration.In a previous paper (Bennekou, P. 1988.J. Membrane Biol. 102:225–234) a kinetic model for the translocation of a protonophore, CCCP, across the human red cell membrane was established. This model accounts for the protonophore reequilibration following abrupt changes in membrane potential.In this paper, the limitations of the method with regard to the estimation of transient membrane potentials are examined, using the transport model to simulate changes in extracellular pH in response to noninstantaneous changes in membrane potential. The temperature and time resolution calculated from the model are reported.Furthermore, it is shown that the transport model established for CCCP is valid for another protonophore, TCS, thus indicating the general validity of the transport scheme for the entire class of protonophores.     

Growth and reproduction of Scytosiphon lomentaria (Fucophyceae) in relation to temperature in two populations from Denmark

Two strains of Scytosiphon lomentaria from Denmark have been grown under long-day conditions on a temperature-gradient plate. The material from north-west Jutland (strain No. 46) has a reduced prostrate system (a few-celled lump) whereas the other material from the Sound (strain No. S41) has a well-developed crust. This difference plays a significant role in growth and reproductive patterns. Strain No. 46 rapidly produces a great number of cylindrical macrothalli. Within a four-week experimental period, formation of swarmers from plurilocular sporangia on the macrothalli takes place and results in a maximum dry weight of 32.9 mg at 18.2°C.
Swarmers from the macrothalli of strain S41 always develop into knot-filamentous or crust-shaped thalli. The development of the macrothalli, therefore, takes a longer time than in strain 46 and the maximum dry weight is only 16.5 mg at 16°C even within an experimental period of six weeks. The reproductive pattern is more complex than in strain 46. The crusts produce unilocular sporangia at temperatures between 16°C and 23.8°C, and function, therefore, as the reproductive system during the summer as macrothalli disappear at temperatures above 18°C both in nature and in culture. Formation of plurilocular sporangia on macrothalli starts when they are from 6–9 weeks old at temperatures between 11°C and 18°C.
The temperature range for growth in the two strains also differs: Strain 46 grows from 7.3–24.4°C while strain S41 grows at least from 4–5.1°C to an upper limit of 27.1°C, but lower limit has not been determined precisely in the present experiment. These results indicate that temperature ecotypes exist, even within a limited geographic area.     

Diversity,taxonomy and autecology of autochtonous epipelic cyanobacteria of the genus <Emphasis Type="Italic">Komvophoron</Emphasis> (Borziaceae,Oscillatoriales): a study on populations from the Czech Republic and British Isles

Epipelic cyanobacteria were studied in bottom sediments from ponds and lakes in the United Kingdom and the Czech Republic, covering a trophic gradient from deep, oligotrophic, glacial lakes to shallow, eutrophic, urban manmade ponds. Although many planktic species were observed, the most frequently occurring cyanobacteria were the motile filamentous genera Oscillatoria, Phormidium, Pseudanabaena and Komvophoron. These genera represent autochtonous epipelic assemblages, occurring in upper surface layers of the sediments throughout the year and exhibiting seasonal variation in their abundancy. The occurrence of individual Komvophoron species was influenced by sediment quality, particularly the proportions of fine mud and organic detritus. A new species Komvophoron hindakii sp. nov. is described. Morphological features, autecology and occurrence of all the Komvophoron-like morphospecies found are discussed.