Growth and reproduction of asexual konjak plants (Amorphophalus konjac K. koch)

To examine the differences of the growth and reproduction of different-aged plants, 0-, 1-, and 2-year plants ofAmorphophalus konjac were investigated. RGR and daily net production per unit productive part, relative net-production rate (α′), of the 0-year plant were largest, although NAR was highest in the 2-year plant. This was due to the large LAR of the 0-year plant, owing to its large SLA. With increase in age, LAR decreased and NAR increased. Thus, it appeared that the age of plant exerts two opposite effects on dry-matter production. Since these effects cancel each other out, differences in RGR and α′ between the two older plants were not significant. We estimated that plant size appears to be primarily responsible for these effects. The 0-year plant showed the least distribution ratio of net production into reproductive (storage) organs, and the highest productivity of the reproductive part. The ratio of the production of corm to total reproductive-part production, the D-reproduction index, was independent of age, and critical size in vegetative propagation could not be detected.     

Extracellular production and affinity purification of recombinant proteins with Escherichia coli using the versatility of the maltose binding protein

Recombinant proteins are essential products of today's industrial biotechnology. In this study we address two crucial factors in recombinant protein production: (i) product accessibility and (ii) product recovery. Escherichia coli, one of the most frequently used hosts for recombinant protein expression, does not inherently secrete proteins into the extracellular environment. The major drawback of this expression system is, therefore, to be found in the intracellular protein accumulation and hampered product accessibility. We have constructed a set of expression vectors in order to facilitate extracellular protein production and purification. The maltose binding protein from E. coli is used as fusion partner for several proteins of interest allowing an export to the bacteria's periplasm via both the Sec and the Tat pathway. Upon coexpression of a modified Cloacin DF13 bacteriocin release protein, the hybrid proteins are released into the culture medium. This essentially applies to a distinguished reporter molecule, the green fluorescent protein, for which an extracellular production was not reported so far. The sequestered proteins can be purified to approximate homogeneity by a simple, rapid and cheap procedure which utilizes the affinity of the maltose binding protein to α-1,4-glucans.     

The importance of ‘tooglies’ to the ethnography of F.E. Williams,government anthropologist

The theoretical orientation, encapsulated in the semi‐serious concept of ‘tooglies'of F.E. Williams, government anthropologist for the Territory of Papua in the inter‐war period, is considered. His ideas about culture change are contrasted with those of Bronislaw Malinowski, who acted as his mentor at one time. His treatment of the social organisational anomaly of Sogeri Koiari ‘sex affiliation’, often cited as a case of parallel bilineal descent, is compared with Margaret Mead's analysis of the ‘Mundugumor ropes’, which is classed as the opposite, cross‐sex bilineal descent. It is shown that Williams was able to get a clearer insight into this anomalous data than Mead and Fortune, and, on the whole, worked with an understanding of culture and Papuan social organisation that presages the relational approach of today.     

Phytoplankton biomass and production in northern South China Sea during summer: Influenced by Pearl River discharge and coastal upwelling

Chlorophyll a and primary production were studied in northern South China Sea during summer from 2007 to 2008. Microplankton dominated total phytoplankton biomass in the coast, while picoplankton dominated in the offshore. Algae bloom caused by Thalassionema nitzschioides was found at the subsurface of upwelling regions (D2, C2) in 2008, and maximum of phytoplankton abundance reached 1.58 × 10⁶ ind L^?1. Integrated primary production ranged from 189.3 to 976.2 mg m^?2 d^?1 in 2007, and ranged from 652.1 to 6601 mg m^?2 d^?1 in 2008. PP showed positive relationship with IPP (p < 0.01) and negative relationship with SST (p < 0.05). Coastal upwelling and Pearl River discharge sustained high PP, and played important role in regulating the phytoplankton biomass and production.     

Identification of residues in the human guanylate-binding protein 1 critical for nucleotide binding and cooperative GTP hydrolysis

The guanylate-binding proteins (GBPs) form a group of interferon-gamma inducible GTP-binding proteins which belong to the family of dynamin-related proteins. Like other members of this family, human guanylate-binding protein 1 (hGBP1) shows nucleotide-dependent oligomerisation that stimulates the GTPase activity of the protein. A unique feature of the GBPs is their ability to hydrolyse GTP to GDP and GMP. In order to elucidate the relationship between these findings, we designed point mutants in the phosphate-binding loop (P-loop) as well as in the switch I and switch II regions of the protein based on the crystal structure of hGBP1. These mutant proteins were analysed for their interaction with guanine nucleotides labeled with a fluorescence dye and for their ability to hydrolyse GTP in a cooperative manner. We identified mutations of amino acid residues that decrease GTPase activity by orders of magnitude a part of which are conserved in GTP-binding proteins. In addition, mutants in the P-loop were characterized that strongly impair binding of nucleotide. In consequence, together with altered GTPase activity and given cellular nucleotide concentrations this results in hGBP1 mutants prevailingly resting in the nucleotide-free (K51A and S52N) or the GTP bound form (R48A), respectively. Using size-exclusion chromatography and analytical ultracentrifugation we addressed the impact on protein oligomerisation. In summary, mutants of hGBP1 were identified and biochemically characterized providing hGBP1 locked in defined states in order to investigate their functional role in future cell biology studies.     

A comparison of nuclear and mitochondrial cline shapes in a hybrid zone in the Sceloporus grammicus complex (Squamata; Phrynosomatidae)

The F5 and FM2 chromosome races of the Sceloporus grammicus complex form a hybrid zone in the Mexican state of Hidalgo. Previous studies of this zone have assessed genetic structure by averaging estimates of shape and width across three diagnostic chromosome markers. This approach is likely to mask subtle differences in cline shape among loci (e.g. selected vs. neutral), and obscure any displacement of cline centres (if present). Here we use maximum likelihood methods to construct the best fitting individual clines for three chromosomal markers, and also add two new markers; the mitochondrial DNA (mtDNA) locus, and the nuclear ribosomal DNA (rDNA) repeat. For each locus, hybrid zone models were fitted by cline shape and width, and the position and number of segments describing the centre of the zone. Pairwise comparisons between all clines revealed concordance between chromosomes 2 and 6, but significant discordance in cline structure among all other paired combinations. The concordance of chromosomes 2 and 6 suggests that these clines are maintained by genome-wide forces. The discordance of the chromosome 1 cline suggests an influence of asymmetric introgression, while the mtDNA cline is probably influenced by selection and drift. The rDNA locus reveals a pattern best explained by either extreme asymmetric introgression or gene conversion. The structure of zone indicates that genome-wide processes and locus specific selective forces as well as drift, are operating to different degrees on different loci. The locus-by-locus approach used here permits a finer discrimination among possible mechanisms responsible for the maintenance of the individual clines.     

A simple method to control the bacterial production of cellulosic films in order to obtain dried pellicles presenting a desired average thickness

During the bacterial production of cellulosic films on non-agitated liquid media, the mass of the system does not change. An equation is therefore proposed to evaluate the volume of inoculated medium to be placed in a given tray so that, when no more free aqueous medium may be detected under the wet pellicle produced, a dried film of a desired average thickness is produced.     

Anthropogenic nitrogen sources and relationships to riverine nitrogen export in the northeastern U.S.A.

Human activities have greatly altered the nitrogen (N) cycle, accelerating the rate of N fixation in landscapes and delivery of N to water bodies. To examine relationships between anthropogenic N inputs and riverine N export, we constructed budgets describing N inputs and losses for 16 catchments, which encompass a range of climatic variability and are major drainages to the coast of the North Atlantic Ocean along a latitudinal profile from Maine to Virginia. Using data from the early 1990's, we quantified inputs of N to each catchment from atmospheric deposition, application of nitrogenous fertilizers, biological nitrogen fixation, and import of N in agricultural products (food and feed). We compared these inputs with N losses from the system in riverine export.The importance of the relative sources varies widely by catchment and is related to land use. Net atmospheric deposition was the largest N source (>60%) to the forested basins of northern New England (e.g. Penobscot and Kennebec); net import of N in food was the largest source of N to the more populated regions of southern New England (e.g. Charles & Blackstone); and agricultural inputs were the dominant N sources in the Mid-Atlantic region (e.g. Schuylkill & Potomac). Over the combined area of the catchments, net atmospheric deposition was the largest single source input (31%), followed by net imports of N in food and feed (25%), fixation in agricultural lands (24%), fertilizer use (15%), and fixation in forests (5%). The combined effect of fertilizer use, fixation in crop lands, and animal feed imports makes agriculture the largest overall source of N. Riverine export of N is well correlated with N inputs, but it accounts for only a fraction (25%) of the total N inputs. This work provides an understanding of the sources of N in landscapes, and highlights how human activities impact N cycling in the northeast region.     

Identifying protein construct variants with increased crystallization propensity--a case study

This study describes an efficient multiparallel automated workflow of cloning, expression, purification, and crystallization of a large set of construct variants for isolated protein domains aimed at structure determination by X-ray crystallography. This methodology is applied to MAPKAP kinase 2, a key enzyme in the inflammation pathway and thus an attractive drug target. The study reveals a distinct subset of truncation variants with improved crystallization properties. These constructs distinguish themselves by increased solubility and stability during a parallel automated multistep purification process including removal of the recombinant tag. High-throughput protein melting point analysis characterizes this subset of constructs as particularly thermostable. Both parallel purification screening and melting point determination clearly identify residue 364 as the optimal C terminus for the kinase domain. Moreover, all three constructs that ultimately crystallized feature this C terminus. At the N terminus, only three amino acids differentiate a noncrystallizing from a crystallizing construct. This study addresses the very common issues associated with difficult to crystallize proteins, those of solubility and stability, and the crucial importance of particular residues in the formation of crystal contacts. A methodology is suggested that includes biophysical measurements to efficiently identify and produce construct variants of isolated protein domains which exhibit higher crystallization propensity.     

Functional expression of human pyruvate carboxylase for reduced lactic acid formation of Chinese hamster ovary cells (DG44)

To investigate the effect of human pyruvate carboxylase (hPC) on lactate formation in Chinese hamster ovary (CHO) cell lines, FLAG-tagged hPC was introduced into a dihydrofolate-deficient CHO cell line (DG44). Three clones expressing high levels of hPC, determined by Western blotting using an anti-FLAG monoclonal antibody, and a control cell line were established. Immunocytochemistry revealed that a substantial amount of expressed hPC protein was localized in the mitochondria of the cells. hPC expression did not impair cell proliferation. Rather, it improved cell viability at the end of adherent batch cultures with the serum-containing medium probably because of reduced lactate formation. Compared with control cells, specific lactate production rate of the three clones was decreased by 21–39%, which was because of a decreased specific glucose uptake rate and yield of lactate from glucose. Reduced lactate formation by hPC expression was also observed in suspension fed-batch cultures using a serum-free medium. Taken together, these results demonstrate that through the expression of the hPC enzyme, lactate formation in CHO cell culture can be efficiently reduced.