Canopy scale measurements of CO2 and water vapor exchange along a precipitation gradient in southern Africa

Short‐term measurements of carbon dioxide, water, and energy fluxes were collected at four locations along a mean annual precipitation gradient in southern Africa during the wet (growing) season with the purpose of determining how the observed vegetation–atmosphere exchange properties are functionally related to the long‐term climatic conditions. This research was conducted along the Kalahari Transect (KT), one in the global set of International Geosphere‐Biosphere Program transects, which covers a north–south aridity gradient, all on a homogenous sand formation. Eddy covariance instruments were deployed on a permanent tower in Mongu, Zambia (879 mm of rainfall per year), as well as on a portable tower in Maun (460 mm yr^?1), Okwa River Crossing (407 mm yr^?1), and Tshane (365 mm yr^?1), Botswana for several days at each site. The relationships between CO₂ flux, F_c, and photosynthetically active radiation were described well by a hyperbolic fit to the data at all locations except for Mongu, the wettest site. Here, there appeared to be an air temperature effect on F_c. While daytime values of F_c routinely approached or exceeded ?20 μmol m^?2 s^?1 at Mongu, the magnitude of F_c remained less than ?10 μmol m^?2 s^?1 when the air temperature was above 27°C. Canopy resistances to water vapor transfer, r_c, displayed an overall decline from the wetter sites to the more arid sites, but the differences in r_c could be almost exclusively accounted for by the decrease in leaf area index (LAI) from north to south along the KT. Ecosystem water use efficiency (WUE), defined as the ratio of net carbon flux to evapotranspiration, showed a general decrease with increasing vapor pressure deficit, D, for all of the sites. The magnitudes of WUE at a given D, however, were dissimilar for the individual sites and were found to be stratified according to the position of the sites along the long‐term aridity gradient. For example, Mongu, which has the wettest climate, has a much lower WUE for like levels of D than Tshane, which historically has the most arid climate. Given the similar inferred stomatal resistances between the sites, the disparate carbon uptake behavior for the grass vs. woody vegetation is the likely cause for the observed differences in WUE along the aridity gradient. The short‐term flux measurements provide a framework for evaluating the vegetation's functional adaptation to the long‐term climate and provide information that may be useful for predicting the dynamic response of the vegetation to future climate change.     

Probing the flexibility of the DsbA oxidoreductase from Vibrio cholerae--a 15N - 1H heteronuclear NMR relaxation analysis of oxidized and reduced forms of DsbA

We have determined the structure of the reduced form of the DsbA oxidoreductase from Vibrio cholerae. The reduced structure shows a high level of similarity to the crystal structure of the oxidized form and is typical of this class of enzyme containing a thioredoxin domain with an inserted alpha-helical domain. Proteolytic and thermal stability measurements show that the reduced form of DsbA is considerably more stable than the oxidized form. NMR relaxation data have been collected and analyzed using a model-free approach to probe the dynamics of the reduced and oxidized states of DsbA. Akaike's information criteria have been applied both in the selection of the model-free models and the diffusion tensors that describe the global motions of each redox form. Analysis of the dynamics reveals that the oxidized protein shows increased disorder on the pico- to nanosecond and micro- to millisecond timescale. Many significant changes in dynamics are located either close to the active site or at the insertion points between the domains. In addition, analysis of the diffusion data shows there is a clear difference in the degree of interdomain movement between oxidized and reduced DsbA with the oxidized form being the more rigid. Principal components analysis has been employed to indicate possible concerted movements in the DsbA structure, which suggests that the modeled interdomain motions affect the catalytic cleft of the enzyme. Taken together, these data provide compelling evidence of a role for dynamics in the catalytic cycle of DsbA.     

Involving undergraduates in the annotation and analysis of global gene expression studies: creation of a maize shoot apical meristem expression database

Through a multi-university and interdisciplinary project we have involved undergraduate biology and computer science research students in the functional annotation of maize genes and the analysis of their microarray expression patterns. We have created a database to house the results of our functional annotation of >4400 genes identified as being differentially regulated in the maize shoot apical meristem (SAM). This database is located at http://sam.truman.edu and is now available for public use. The undergraduate students involved in constructing this unique SAM database received hands-on training in an intellectually challenging environment, which has prepared them for graduate and professional careers in biological sciences. We describe our experiences with this project as a model for effective research-based teaching of undergraduate biology and computer science students, as well as for a rich professional development experience for faculty at predominantly undergraduate institutions.     

Diffusible signal factor-dependent cell-cell signaling and virulence in the nosocomial pathogen Stenotrophomonas maltophilia

The genome of Stenotrophomonas maltophilia encodes a cell-cell signaling system that is highly related to the diffusible signal factor (DSF)-dependent system of the phytopathogen Xanthomonas campestris. Here we show that in S. maltophilia, DSF signaling controls factors contributing to the virulence and antibiotic resistance of this important nosocomial pathogen.     

Microwave Synthesis of Prion Protein Fragments up to 111 Amino Acids in Length Generates Biologically Active Peptides

Misfolded conformers of the prion protein are aetiologically implicated in neurodegenerative conditions termed prion diseases (also known as transmissible spongiform encephalopathies). Two constitutively expressed N-terminal peptides corresponding to human residues 23–90 and 23–111 are thought to serve normal physiological roles related to neuronal protection with membrane binding possibly playing a part in their mechanism of action. These peptides, along with several derivatives up to 111 residues in length, have been produced by microwave assisted peptide synthesis. HPLC and MS characterisation showed that the peptides were manufactured in good yields at high purity. Peptides were assayed by fluorescence spectroscopy for synthetic lipid-membrane binding activity and by dichlorodihydrofluorescein diacetate assay for the amelioration of reactive oxygen species production. Results of these assays were similar to those reported for the wild type recombinant PrP, demonstrating that these synthetic peptides are useful for biological and chemical assays of PrP activity. Further, the longest peptide 1–111 was dimerised via a single internal cystine residue with good yield. The high yields and low purification burden of the microwave assisted synthesis method lends itself to the production of difficult to produce peptides for such studies.     

Quantifying and resolving multiple vector transformants in S. cerevisiae plasmid libraries

Background

Two sequential enzymes in the production of sialic acids, N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), were overexpressed as double-tagged gene fusions. Both were tagged with glutathione S-transferase (GST) at the N-terminus, but at the C-terminus, one was tagged with five contiguous aspartate residues (5D), and the other with five contiguous arginine residues (5R).

Results

Both fusion proteins were overexpressed in Escherichia coli and retained enzymatic activity. The fusions were designed so their surfaces were charged under enzyme reaction conditions, which allowed isolation and immobilization in a single step, through a simple capture with either an anionic or a cationic exchanger (Sepharose Q or Sepharose SP) that electrostatically bound the 5D or 5R tag. The introduction of double tags only marginally altered the affinity of the enzymes for their substrates, and the double-tagged proteins were enzymatically active in both soluble and immobilized forms. Combined use of the fusion proteins led to the production of N-acetyl-D-neuraminic acid (Neu5Ac) from N-acetyl-D-glucosamine (GlcNAc).

Conclusion

Double-tagged gene fusions were overexpressed to yield two enzymes that perform sequential steps in sialic acid synthesis. The proteins were easily immobilized via ionic tags onto ionic exchange resins and could thus be purified by direct capture from crude protein extracts. The immobilized, double-tagged proteins were effective for one-pot enzymatic production of sialic acid.     

Parent‐Child Feeding Strategies and Their Relationships to Child Eating and Weight Status

Parental feeding styles may promote overeating or overweight in children. A comprehensive literature review was undertaken to summarize the associations between parental feeding styles and child eating and weight status. Twenty‐two studies were identified. We systematically coded study attributes and outcomes and tested for patterns of association. Nineteen studies (86%) reported at least one significant association between parental feeding style and child outcome, although study methodology and results varied considerably. Studies measuring parental feeding restriction, as opposed to general feeding control or another feeding domain, were more likely to report positive associations with child eating and weight status. Certain associations differed by gender and by outcome measurement (e.g., rate of eating as opposed to total energy intake). Parental feeding restriction, but no other feeding domain, was associated with increased child eating and weight status. Longitudinal studies are needed to test underlying causal pathways, including bidirectional causal models, and to substantiate findings in the presence of other obesity risk factors.     

Identification and Preliminary Characterization of a Ca2+- Dependent High-Affinity Binding Site for Inositol-1,4,5-Trisphosphate from Chenopodium rubrum

Using a radioligand-binding assay we have identified a Ca2+- dependent high-affinity D-myo-inositol-1,4,5-trisphosphate (InsP3) binding site in a membrane vesicle preparation from Chenopodium rubrum. Millimolar concentrations of Ca2+ were required to observe specific binding of [3H]InsP3. A stable equilibrium between bound and free ligand was established within 5 min and bound [3H]InsP3 could be completely displaced by InsP3 in a time- and concentration-dependent manner. Displacement assays indicated a single class of binding sites with an estimated dissociation constant of 142 [plus or minus] 17 nM. Other inositol phosphates bound to the receptor with much lower affinity. The glycosaminoglycan heparin was an effective competitor for the binding site (inhibitor concentration for 50% displacement = 534 nM). ATP at higher, although physiologically relevant, concentrations (inhibitor concentration for 50% displacement = 241 [mu]M) also displaced [3H]InsP3 from the receptor. Recent studies in animals have highlighted the importance of Ca2+ regulation of InsP3-induced Ca2+ release. The potential for the operation of similar regulatory mechanisms in plants is discussed.