Loss and recovery of protein productivity in genetically modified plant cell lines

This paper addresses the problem of decreasing protein expression levels in genetically modified plant cells. A modeling approach was used to explain the loss of productivity over successive generations. Using this model, productivity losses were simulated for two cell lines. Although the lines were relatively stable, the protein production level could decrease by more than 80% after a large number of generations. Motivated by this problem, a dispersion method was developed to isolate productive cells from existing cell suspensions. Dilution of transgenic cells in a feeder layer of nonproducing cells at a ratio of 1:1000 facilitated the recovery of distinct, separate daughter colonies. Applying this method, it was possible to recover high producing cell lines.     

Changes in Oregon white oak (Quercus garryana Dougl. ex Hook.) following release from overtopping conifers

Oregon white oak or Garry oak (Quercus garryana Dougl. ex Hook.) is a shade-intolerant, deciduous species that has been overtopped by conifers during the past century in parts of its range due to an altered disturbance regime. We examined the response of suppressed Oregon white oak trees in western Washington, USA, to three levels of release from overtopping Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco). We treated individual oak trees with either full release from competition, partial (“half”) release from competition, or a stand-level thinning of Douglas-fir not directed toward release (control). Five years after treatment, oak trees had suffered no mortality or windthrow. Stem diameter growth was 194% greater in the full-release treatment relative to the control. Acorn production varied widely by year, but in years of higher production, acorn production was significantly greater in both release treatments than in the control. Frequency of epicormic branch formation was significantly increased for years 1 and 2 by the full release; the greatest response occurred between 2 and 6 m above ground level. The greatest number of epicormic branches formed on trees on which the majority of original limbs had died back prior to treatment. Trees with relatively less crown dieback at the time of treatment generally had greater stem growth and acorn production responses to release treatments. Our findings indicate that these released Oregon white oak trees are beginning to recover after an extended period of suppression.     

A re-analysis of agricultural production and consumption: implications for understanding the British Iron Age

Patterning in the carbonized seed assemblages from Iron Age sites in Britain has led to the development of several explanatory models. The most influential of these, by Martin Jones, proposed that grain-rich assemblages represent producer sites and weed-/chaff-rich assemblages consumer sites. The assumptions underlying this model and the method of constructing the diagrams are discussed and a new approach is put forward, stressing the need for appropriate levels of analysis and interpretation. It is concluded that a predominance of grain-rich samples is far more likely to be an indicator of the scale of production and consumption, than a means of distinguishing between the two. A review of the evidence from Iron Age Britain indicates that grain-rich site assemblages primarily occur in the south of the country, and frequently co-occur with pits, used for the storage of surplus grain. Moreover, such pits are concentrated in hillforts. It is proposed that the grain stored in such pits may have been used in large communal feasts and that the hillforts functioned as locations for feasting.

Human impact during the Bronze Age on the vegetation at Lago Lucone (northern Italy)

Lake-sediment records were used to reconstruct human impact on the landscape around Lago Lucone (45°33′N, 10°29′E, 249 m a.s.l.), a former lake in the western amphitheatre system of the Lago di Garda. Presence of prehistoric human populations is attested by pile-dwelling settlements from the Early-Middle Bronze Age, with one settlement at a distance of only 100 m from the coring site. Pollen, plant-macrofossil and microscopic charcoal analyses were applied to a 250 cm sediment core with four dates providing the time control. A mixed oak forest that was important during the Early-Middle Holocene was cleared and replaced by open vegetation during the Bronze Age (∼2000–1100 b.c.) when open lands were estimated to have covered more than 60% of the total relevant pollen-source area. During a phase of high human impact, independent climatic proxies suggest warm and dry climatic conditions. Later, ca. 1100 b.c., palaeobotanical evidence indicates a sharp decrease in human pressure in the Lago Lucone area. The comparison with other sedimentary palaeocultural records shows that the period 1300–1100 b.c. was characterised by general declines of agricultural activities both south and north of the Alps. These declines have been previously attributed to a change towards wetter and colder climatic conditions in and around the Alps. However, the decline in human impact around Lago Lucone cannot be exclusively attributed to climatic variation. Therefore other forcing factors independent of climatic changes, such as cultural crises or changes in spatial organisation of the habitats, cannot be ruled out under the present state of knowledge.     

Influence of maternal dietary calcium levels on milk zinc, calcium and phosphorus contents and milk production in rats

The aim of this study was to analyze zinc (Zn), calcium (Ca) and phosphorus (P) contents in milk and the lactational performance in rats fed different Ca levels. Female Wistar rats were fed during pregnancy and lactation with experimental diets containing 20% protein and high (0.90%, HCa), normal (0.60%, NCa) or low (0.20%, LCa) Ca levels. Milk samples were collected after 15 days to determine the milk mineral composition. Pup weight was recorded from birth to weaning (litter size: 6-8 pups) to determine weight gain and calculate milk production. At delivery there were no significant differences in the body weight of the pups between the groups, but at day 15, the LCa group showed lower values than both NCa and HCa groups (p<0.05). The weight gain of the LCa group was significantly lower than of the HCa and NCa groups, between delivery and day 5 (p<0.05). This reduced rate of weight gain led to the LCa group reaching weaning weight later than the other groups. Milk production (g/pup/day) was significantly lower when dams were fed the LCa than the NCa and HCa diets (p<0.05). There were no significant differences among the groups in milk Ca, P and Zn levels and Ca/P ratio. The body mineral composition of the pups at birth did not differ between the groups; at weaning, however, both LCa and HCa groups had lower element contents than the NCa group (p<0.05). In conclusion, dams fed with a diet containing low Ca levels produced smaller volumes of milk and their pups reached weaning weights later than the other groups. As the milk mineral composition was not affected, it can be hypothesized that in dams fed low dietary Ca, the smaller milk yield might have been a way of maintaining milk quality. High Ca levels affected neither pregnancy outcome nor lactational performance.     

Screening and characterization of yeasts for xylitol production

AIMS: To discover novel naturally occurring xylitol producing yeast species with potential for industrial applications. METHODS AND RESULTS: Exactly 274 strains were cultivated on both solid and liquid screening medium with xylose as the sole carbon resource. Five strains were selected on the basis of significant growth and high degree of xylose assimilation. Their phylogenetic position was confirmed by the PCR-RFLP and sequence analysis of the D1/D2 domain of the 5' end of the large subunit rDNA gene (5'-LSU rDNA). Enzymatic analysis was conducted to compare xylose metabolism in each strain. Candida guilliermondii Xu280 and Candida maltosa Xu316 were found to have high xylose consumption rates and xylitol yields in the batch fermentation under micro-aerobic condition. The effect of the different media with high initial xylose concentration on biosynthesis of xylitol by both strains was investigated. CONCLUSIONS: We have identified Candida spp. strains, which exhibit high levels of xylitol production from xylose suggesting that these may have potential for industrial applications. SIGNIFICANCE AND IMPACTS OF THE STUDY: Microbial species are of importance for xylitol production. Xylitol production involves complicated metabolic regulation including xylose transport, production of key enzymes and cofactor regeneration. Thus, screening of naturally occurring xylose-utilizing micro-organisms is a viable and effective mean to obtain xylitol producing organisms with industrial application. Moreover, the research on selected strains will contribute to a better understanding of regulatory properties of xylose metabolism in different yeasts.     

Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase that increases the production rate of D-tagatose

AIMS: Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase used to increase the production rate of D-tagatose. METHODS AND RESULTS: A mutated gene was obtained by an error-prone polymerase chain reaction using L-arabinose isomerase gene from G. stearothermophilus as a template and the gene was expressed in Escherichia coli. The expressed mutated L-arabinose isomerase exhibited the change of three amino acids (Met322-->Val, Ser393-->Thr, and Val408-->Ala), compared with the wild-type enzyme and was then purified to homogeneity. The mutated enzyme had a maximum galactose isomerization activity at pH 8.0, 65 degrees C, and 1.0 mM Co2+, while the wild-type enzyme had a maximum activity at pH 8.0, 60 degrees C, and 1.0-mM Mn2+. The mutated L-arabinose isomerase exhibited increases in D-galactose isomerization activity, optimum temperature, catalytic efficiency (kcat/Km) for D-galactose, and the production rate of D-tagatose from D-galactose. CONCLUSIONS: The mutated L-arabinose isomerase from G. stearothermophilus is valuable for the commercial production of D-tagatose. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes knowledge on the characterization of a mutated L-arabinose isomerase, and allows an increased production rate for D-tagatose from D-galactose using the mutated enzyme.     

Proteases production by two Vibrio species on residuals marine media

A comparative study was carried out on the growth and production of alkaline proteases by two Vibrio species using different marine peptones from fish viscera residues. The bacteria tested, Vibrio anguillarum and Vibrio splendidus, are producers of high levels of proteolytic enzymes which act as factors of virulence in fish cultures, causing high mortality rates. The kinetic assays and subsequent comparison with the parameters obtained from the adjustment to various mathematical models, highlighted the potential interest of the media formulated, for their possible production on an industrial scale, particularly the production of proteases by V. anguillarum growing in rainbow trout and squid peptones.     

Isolation and characterization of two novel ethanol-tolerant facultative-anaerobic thermophilic bacteria strains from waste compost

In a search for potential ethanologens, waste compost was screened for ethanol-tolerant thermophilic microorganisms. Two thermophilic bacterial strains, M5EXG and M10EXG, with tolerance of 5 and 10% (v/v) ethanol, respectively, were isolated. Both isolates are facultative anaerobic, non-spore forming, non-motile, catalase-positive, oxidase-negative, Gram-negative rods that are capable of utilizing a range of carbon sources including arabinose, galactose, mannose, glucose and xylose and produce low amounts of ethanol, acetate and lactate. Growth of both isolates was observed in fully defined minimal media within the temperature range 50–80°C and pH 6.0–8.0. Phylogenetic analysis of the 16S rDNA sequences revealed that both isolates clustered with members of subgroup 5 of the genus Bacillus. G+C contents and DNA–DNA relatedness of M5EXG and M10EXG revealed that they are strains belonging to Geobacillus thermoglucosidasius. However, physiological and biochemical differences were evident when isolates M5EXG and M10EXG were compared with G. thermoglucosidasius type strain (DSM 2542^T). The new thermophilic, ethanol-tolerant strains of G. thermoglucosidasius may be candidates for ethanol production at elevated temperatures.     

Screening strains for directed biosynthesis of β-d-mono-glucuronide-glycyrrhizin and kinetics of enzyme production

One fungus, tentatively named Penicillium sp. Li-3, was screened to biosynthesize β-d-mono-glucuronide-glycyrrhizin (GAMG), directly. Using glycyrrhizin as elicitor and the sole carbon source, this strain was capable of expressing β-d-glucuronidase, one intracellular enzyme with high substrate specificity. And glycyrrhizin was hydrolyzed directly into GAMG by enzyme from Penicillium sp. Li-3 with high production. It was found that the mol conversion ratio of this reaction was up to 88.45%. Research about kinetics of β-d-glucuronidase production showed that the cell growth and enzyme production of this strain was partial coupled. During the expressing of target enzyme, carbon catabolite repression existed, so only glycyrrhizin was the best carbon source as well as the elicitor. It was found that the surfactant (Tween 80 0.12%) could improve the ability of enzyme production markedly. Under the condition of initial pH 4.8 of the medium and 32 °C of the culture temperature, the maximum enzyme activity of 181.53 U ml⁻¹ was obtained.