Characterization of liposomes coated with S-layer proteins from lactobacilli

The stability of liposomes coated with S-layer proteins from Lactobacillus brevis and Lactobacillus kefir was analyzed as a previous stage to the development of a vaccine vehicle for oral administration. The interactions of the different S-layer proteins with positively charged liposomes prepared with soybean lecithin or dipalmitoylphosphatidylcholine were studied by means of the variation of the Z potential at different protein-lipid ratios, showing that both proteins were able to attach in a greater extent to the surface of soybean lecithin liposomes. The capacity of these particles to retain carboxyfluorescein or calcein by exposure to bile salts, pancreatic extract, pH change and after a thermal shock showed that both S-layer proteins increased the stability of the liposomes in the same magnitude. The non-glycosylated protein from L. brevis protects more efficiently the liposomes at pH 7 than those from L. kefir even without treatment with glutaraldehyde.     

Biochemical acclimation of metabolic enzymes in response to lowered temperature in tadpoles of Limnodynastes peronii

We measured the rate at which the metabolic enzymes lactate dehydrogenase (LDH), citrate synthase (CS), and cytochrome c oxidase (CCO) acclimate in the response to lowered temperature in the axial muscle of tadpoles of Limnodynastes peronii (Anura: Myobatrachidae) over 6 weeks. In addition, we measured growth rates of the tadpoles kept at both temperatures and examined the activities of these enzymes in the liver tissue of the control group and cold-acclimated group at the end of the experiment. We found that LDH acclimates in axial muscle; the differences between the control and cold-acclimated group became apparent after 21 days. After 42 days, the activity of LDH in axial muscle in the cold-acclimated group was 30% greater than the control group. Growth rates were maintained at 0.7 mm/week within both treatments despite the 10 degrees C difference in temperature between experimental groups. Both LDH and CS were increased in activity in the liver (5 and 1.3 times greater, respectively, in the cold-acclimated group). The thermal sensitivity (Q(10)) of LDH was between 20 and 30 degrees C in the cold-acclimated group (1.2+/-0.01) when compared to the control group (1.6+/-0.15). The rate at which acclimation in this species occurs is appropriate for seasonal changes in temperature, and these animals may not be able to respond to a rapid drop in temperature.     

Development of a new vitrification solution, VSL, and its application to the cryopreservation of gentian axillary buds

Vitrification methods are convenient for cryopreserving plant specimens, as the specimens are plunged directly into liquid nitrogen (LN) from ambient temperatures. However, tissues and species with poor survival are still not uncommon. The development of vitrification solutions with high survival that cover a range of materials is important. We attempted to develop new vitrification solutions using bromegrass cells and found that VSL, comprising 20% (w/v) glycerol, 30% (w/v) ethylene glycol, 5% (w/v) sucrose, 10% (w/v) DMSO and 10 mM CaCl₂, gave the highest survival following cryopreservation, as determined by fluorescein diacetate staining. However, the cryopreserved cells showed little regrowth, for unknown reasons. To check its applicability, VSL was used to cryopreserve gentian axillary buds and the performance was compared with those of conventional vitrification solutions. Excised gentian stem segments with axillary buds (shoot apices) were two-step precultured with sucrose to induce osmotic tolerance prior to cryopreservation. Gentian axillary buds cryopreserved using VSL following the appropriate preculturing approach exhibited 78% survival (determined by the regrowth capacity), which was comparable to PVS2 and PVS1 and far better than PVS3. VSL had a wider optimal incubation time (20–45 min) than PVS2 and was more suitable for cryopreserving gentian buds. The optimal duration of the first step of the preculture was 7–11 days, and preculturing with sucrose and glucose gave a much higher survival than fructose and maltose. VSL was able to vitrify during cooling to LN temperatures, as glass transition and devitrification points were detected in the warming profiles from differential scanning calorimetry. VSL and its derivative, VSL+, seem to have the potential to be good alternatives to PVS2 for the cryopreservation of some materials, as exemplified by gentian buds. Mitsuteru Suzuki, Pramod Tandon and Masaya Ishikawa contributed equally to the work.     

Statistical inference for serial dilution assay data

Serial dilution assays are widely employed for estimating substance concentrations and minimum inhibitory concentrations. The Poisson-Bernoulli model for such assays is appropriate for count data but not for continuous measurements that are encountered in applications involving substance concentrations. This paper presents practical inference methods based on a log-normal model and illustrates these methods using a case application involving bacterial toxins.     

Holocene vegetation history of the Sahel: pollen, sedimentological and geochemical data from Jikariya Lake, north-eastern Nigeria

Aim This study aims to separate regional and local controls on Holocene vegetation development and examine how well pollen records reflect climate change in a semi‐arid region. The relative importance of climate and human activity as agents of vegetation change in the Sahel during the late Holocene is also considered. Location Jikariya Lake, an inter‐dune depression in the Manga Grasslands of north‐eastern Nigeria. Methods Pollen and charcoal were used to provide a record of Holocene vegetation history. Palaeoclimate and hydrological changes were reconstructed from sedimentary and geochemical data. Regional and local influences were separated by comparing the evidence obtained from Jikariya Lake with previously published data from the Manga Grasslands. Results The Manga Grasslands experienced a prolonged wet period during the early and mid‐Holocene, during which swamp forest vegetation with Guinean affinities (Alchornea, Syzygium, Uapaca) occupied the inter‐dune depressions. However, variation in the pollen records between sites suggests that their establishment was dependent on conditions being locally favourable, rather than being directly coupled to regional climate. The pollen records from the Manga Grasslands are more consistent in suggesting the colonization of the dunefields by trees associated with Sudanian savanna (Combretaceae, Detarium) c. 8700 cal. yr bp . The Jikariya Lake pollen data are in accordance with the sedimentological and geochemical data from the region in indicating that the onset of arid conditions occurred progressively during the late Holocene (from c. 4700 cal. yr bp ). Abrupt changes in pollen stratigraphy, recorded at other Manga Grasslands sites 3500 cal. yr bp , appear to be the product of the local passing of ecological thresholds. The dunefield vegetation (Sahelian savanna) appears to have been resilient to (or at least palynologically silent regarding) to the climatic variability of the late Holocene. Main conclusions While climate appears to have been the primary control on vegetation development in the Manga Grasslands during the Holocene, local conditions (particularly depression size and sand influx) had a strong influence on the timing of pollen stratigraphic changes. Anthropogenic influences are difficult to detect, even during the late Holocene.     

Wood density as a proxy of drought-induced forest dieback in silver fir

Drought-induced dieback is a matter of global concern. It has been widely reported and could compromise the climate warming mitigation potential of forests. That is why we need reliable early-warning proxies to achieve better forecasts of forest dieback and tree death. Tree-ring data can provide some of these needed proxies. Here I propose considering minimum (MND) and maximum (MXD) wood density values as proxies of tree vulnerability against drought. This hypothesis is evaluated in silver fir (Abies alba) forests from the western Spanish Pyrenees showing ongoing dieback processes after the severe 1985 drought. MXD increased in response to warm summer conditions in 1985 whereas MND increased in response to growing-season water deficit in 1986. The average between prior MXD (MXD_t-1) and subsequent MND (MND_t) could be used to detect severe drought impacts and forecast dieback.     

Probabilistic reconstruction of ancestral protein sequences

Using a maximum-likelihood formalism, we have developed a method with which to reconstruct the sequences of ancestral proteins. Our approach allows the calculation of not only the most probable ancestral sequence but also of the probability of any amino acid at any given node in the evolutionary tree. Because we consider evolution on the amino acid level, we are better able to include effects of evolutionary pressure and take advantage of structural information about the protein through the use of mutation matrices that depend on secondary structure and surface accessibility. The computational complexity of this method scales linearly with the number of homologous proteins used to reconstruct the ancestral sequence.     

Effects of temperature changes on thymidine kinase in heat- and cold-sensitive cell-cycle mutants and ‘wild-type’ murine P-815 cells

Two heat-sensitive (arrested in G1 at 39.5°C) and two cold-sensitive (arrested in G1 at 33°C) clonal cell-cycle mutants that had been isolated from the same clone (K 21), of the murine mastocytoma P-815 cell line, were tested for thymidine kinase (EC 2.7.1.21) activity. After shift of mutant cells to the nonpermissive temperature, thymidine kinase activity decreased, and minimal levels (i.e., less than 3% of those observed for ‘wild-type’ K 21 cells at the respective temperature) were attained within 16 h in heat-sensitive and after 3–4 days in cold-sensitive mutants, which is in good agreement with kinetics of accumulation of heat-sensitive and cold-sensitive cells in G1 phase. After return of arrested mutant cells to the permissive temperature, thymidine kinase of heat-sensitive cells increased rapidly and in parallel with entry of cells into the S phase. In cultures of cold-sensitive cells, however, initiation of DNA synthesis preceded the increase of thymidine kinase activity by approx. one cell-cycle time. Thymidine kinase activities in revertants of the heat-sensitive and cold-sensitive mutants were similar to those of ‘wild-type’ cells. In ‘wild-type’ K 21 cells incubated at 39.5°C, thymidine kinase activity was approx. 30% of that at 33°C. This difference is attributable, at least in part, to a higher rate of inactivation of the enzyme at 39.5°C, as determined in cultures incubated with cycloheximide. The rapid increase of thymidine kinase activity that occurred after shift of K 21 cells and of arrested heat-sensitive mutant cells from 39.5°C to 33°C was inhibited by actinomycin D and cycloheximide.     

Phospholipid characteristics and neutral lipid fatty acid composition related to temperature and nutritional conditions in ecologically important amphipod species from the northern Baltic Sea

Lipid composition of the benthic, stenothermal amphipod Monoporeia affinis collected from constantly cold deep-water (> 80 m) regions of the northern Baltic Sea was analysed in detail to study phospholipid characteristics, its relation to thermal adaptation as well as potential effects of food quality and seasonal variability. Similar measurements were performed on the littoral, eurythermic amphipod Gammarus spp. Fatty acid (FA) composition of storage lipids of Pontoporeia femorata, a sibling species of M. affinis, was also studied. Interannual and interspecies variability in selected FA was observed both in triacylglycerols (TAG) and phospholipids (PL). Differences in monounsaturated vs. polyunsaturated (MUFA/PUFA) FA combinations of phosphatidyl ethanolamine (PE) diacyl and alkylacyl subgroups were also observed. Seasonal variability in M. affinis was considerably lesser compared to Gammarus spp. A literature synopsis shows that in diacyl PE the share of the FA combination MUFA/PUFA increases with lowering body temperature (40-55% in cold-adapted organisms vs. 5-15% in warm adapted ones), signifying that this characteristic is probably in key position concerning regulation of membrane fluidity in temperature adaptation. According to this feature M. affinis belongs to the group of cold-adapted stenothermic species (Group 1) while eurythermic Gammarus spp. settles in the Group 2 (cold-acclimatized) or 3 (warm-acclimatized/tropical) depending on the status of temperature adaptation. Omnivory and/or periodical food item switching is an important strategy in benthic organisms in high-latitude areas characterised by recurring long poor-nutritional periods. Since many benthic species utilise a time-averaged, integrated food source from phytal and animal matter from various sources the FA composition of TAG of the amphipod species measured here do not exclusively point towards any specific feeding mode or food source. In general, using selected FA as food source markers to assess the diet of field collected more-or-less omnivoric species cannot be considered as an optimal approach. The current study gives more insight to the biochemistry of biological membranes of aquatic crustaceans that is essential in estimation of the capacity of the thermal adaptation/acclimatization of organisms as well as the potential effects of food quality on storage lipids.