Assembly of plant ferredoxin-NADP+ oxidoreductase in Escherichia coli requires GroE molecular chaperones.

We have recently reported the expression in Escherichia coli of an enzymatically competent ferredoxin-NADP+ oxidoreductase from cloned pea genes encoding either the mature enzyme or its precursor protein (Ceccarelli, E. A., Viale, A. M., Krapp, A. R., and Carrillo, N. (1991) J. Biol. Chem. 266, 14283-14287). Processing to the mature form by bacterial protease(s) and FAD assembly occurred in the bacterial cytosol. Expression of ferredoxin-NADP+ reductase in chaperonin-deficient (groE-) mutants of E. coli resulted in partial reductase assembly at permissive growth temperatures (i.e. 30 degrees C), and in total breakdown of holoenzyme assembly, and accumulation as aggregated inclusion bodies at non-permissive temperatures (i.e. 42 degrees C). Coexpression in these mutants of a cloned groESL operon from the phototrophic bacterium Chromatium vinosum resulted in partial or total recoveries of ferredoxin-NADP+ reductase assembly. The overall results indicate that bacterial chaperonins are required for the productive folding/assembly of eucaryotic ferredoxin-NADP+ reductase expressed in E. coli.     

Identification of a Bifunctional Maize C- and O-Glucosyltransferase

Flavonoids accumulate in plant vacuoles usually as O-glycosylated derivatives, but several species can also synthesize flavonoid C-glycosides. Recently, we demonstrated that a flavanone 2-hydroxylase (ZmF2H1, CYP93G5) converts flavanones to the corresponding 2-hydroxy derivatives, which are expected to serve as substrates for C-glycosylation. Here, we isolated a cDNA encoding a UDP-dependent glycosyltransferase (UGT708A6), and its activity was characterized by in vitro and in vivo bioconversion assays. In vitro assays using 2-hydroxyflavanones as substrates and in vivo activity assays in yeast co-expressing ZmF2H1 and UGT708A6 show the formation of the flavones C-glycosides. UGT708A6 can also O-glycosylate flavanones in bioconversion assays in Escherichia coli as well as by in vitro assays with the purified recombinant protein. Thus, UGT708A6 is a bifunctional glycosyltransferase that can produce both C- and O-glycosidated flavonoids, a property not previously described for any other glycosyltransferase.     

Diurnal, circadian and photic regulation of calcium/calmodulin-dependent kinase II and neuronal nitric oxide synthase in the hamster suprachiasmatic nuclei

Mammalian circadian rhythms are entrained by light pulses that induce phosphorylation events in the suprachiasmatic nuclei (SCN). Ca²⁺-dependent enzymes are known to be involved in circadian phase shifting. In this paper, we show that calcium/calmodulin-dependent kinase II (CaMKII) is rhythmically phosphorylated in the SCN both under entrained and free-running (constant dark) conditions while neuronal nitric oxide synthase (nNOS) is rhythmically phosphorylated in the SCN only under entrained conditions. Both p-CaMKII and p-NOS (specifically phosphorylated by CaMKII) levels peak during the day or subjective day. Light pulses administered during the subjective night, but not during the day, induced rapid phosphorylation of both enzymes. Moreover, we found an inhibitory effect of KN-62 and KN-93, both CaMKII inhibitors, on light-induced nNOS activity and nNOS phosphorylation respectively, suggesting a direct pathway between both enzymes which is at least partially responsible of photic circadian entrainment.     

High-Andean vegetation and environmental gradients in northwestern Patagonia,Argentina

Abstract. We analysed the heterogeneity of high-elevation vegetation on three mountains along a west-east transect at 41 °S lat. in the Andes of northwestern Patagonia, Argentina. In this area, high-Andean vegetation occurs as islands on mountain tops above Nothofagus pumilio forests ? with the timberline at ca. 1700 m a.s.l. We recorded floristic, topographic and substrate data in 166 sites stratified according to longitude, altitude, slope and aspect. Vegetation data were classified with TWINSPAN and ordinated with Detrended Correspondence Analysis. The relationship between environmental and floristic variation was analysed using Canonical Correspondence Analysis. In order of importance, geographical longitude, altitude and aspect were the major determinants of vegetation variation, whereas substrate texture and slope appeared less important. The combination of these factors resulted in two main vegetation gradients. The first gradient is related to a moisture availability gradient, primarily determined by longitude and secondarily to variation in wind exposure (east vs. west aspect). The second vegetation gradient is related to variation in temperature, primarily determined by altitude, and secondarily by variation in insolation related to the contrast between north and south aspects. The four communities obtained with TWINSPAN are therefore associated with four characteristic habitat types: moist-cold, moist-warm, dry-cold and dry-warm. The community of warm and dry environments is the richest and has elements in common with dry steppe communities situated at lower elevations to the east, while the vegetation of the cold-moist habitat type has unique elements that are typical of the southern Andes. Although current climatic factors appear to be the major determinants of high-Andean vegetation gradients, historical events of Pleistocene times probably also affected the vegetation patterns we see today.     

Variability of the microbial community in the western Antarctic Peninsula from late fall to spring during a low ice cover year

Although winter conditions play a major role in determining the productivity of the western Antarctic Peninsula (WAP) waters for the following spring and summer, a few studies have dealt with the seasonal variability of microorganisms in the WAP in winter. Moreover, because of regional warming, sea-ice retreat is happening earlier in spring, at the onset of the production season. In this context, this study describes the dynamics of the marine microbial community in the Melchior Archipelago (WAP) from fall to spring 2006. Samples were collected monthly to biweekly at four depths from the surface to the aphotic layer. The abundance and carbon content of bacteria, phytoplankton and microzooplankton were analyzed using flow cytometry and inverted microscopy, and bacterial richness was examined by PCR–DGGE. As expected, due to the extreme environmental conditions, the microbial community abundance and biomass were low in fall and winter. Bacterial abundance ranged from 1.2 to 2.8 × 10⁵ cells ml^?1 showing a slight increase in spring. Phytoplankton biomass was low and dominated by small cells (<2 μm) in fall and winter (average chlorophyll a concentration, Chl-a, of, respectively, 0.3 and 0.13 μg l^?1). Phytoplankton biomass increased in spring (Chl-a up to 1.13 μg l^?1), and, despite potentially adequate growth conditions, this rise was small and phytoplankton was still dominated by small cells (2–20 μm). In addition, the early disappearing of sea-ice in spring 2006 let the surface water exposed to ultraviolet B radiations (UVBR, 280–320 nm), which seemed to have a negative impact on the microbial community in surface waters.     

Carbohydrate metabolism and fruit quality are affected in frost-exposed Valencia orange fruit

Exposure of citrus fruit to frost often results in the development of freeze injury during their maturation in planta. This work was aimed to analyze changes in the biochemistry and enzymology of carbohydrate metabolism in freeze-injured orange fruit ( Citrus sinensis var. Valencia late) and the involvement of oxidative stress in frost damage. The activities of pyrophosphate-dependent phosphofructokinase, phosphoenolpyruvate carboxylase and several fermentative enzymes increased in frost-exposed (FE) fruit, while NADP-malic enzyme and the mitochondrial isoform of NAD-malate dehydrogenase showed a reduction in their activities. Western blot analysis indicated a correlation between activity levels and protein content. Respiration rate in whole fruit was reduced by 40%, whereas the flavedo showed a more pronounced decline (53%). Volatile compound (i.e. ethanol and acetaldehyde) content was significantly higher in FE fruit than in control, as was that of l -malate (three-fold). Additionally, FE fruit showed a marked decrease in the maturity index (24%) because of a higher titratable acidity (39%). Evidence is presented that oxidative stress is involved in freeze-induced damage of orange fruit, where oxidative damage to lipids and proteins, and a greater electrolyte leakage in the flavedo were also observed. The results suggest that freezing temperatures provoke a notable metabolic switch in citrus fruit toward a fermentative stage, resulting in low-quality fruits.     

Timeliness of Clinic Attendance Is a Good Predictor of Virological Response and Resistance to Antiretroviral Drugs in HIV-Infected Patients

Background

Ensuring long-term adherence to therapy is essential for the success of HIV treatment. As access to viral load monitoring and genotyping is poor in resource-limited settings, a simple tool to monitor adherence is needed. We assessed the relationship between an indicator based on timeliness of clinic attendance and virological response and HIV drug resistance.

Methods

Data from 7 virological cross-sectional studies were pooled. An adherence indicator was calculated as the number of appointments attended with delay divided by the number of months between antiretroviral treatment (ART) initiation and date of virological testing and multiplying this by 100. Delays of 1 or more to 5 or more days were considered in turn. Multivariate random-intercept logistic regression was fitted to examine the effect on outcomes, separately for adults and children.

Results

A total of 3580 adults and 253 children were included. Adults were followed for a median of 26.0 months (IQR 12.8-45.0) and attended a median of 24 visits (IQR 13–34). The 1-day delay adherence indicator was strongly associated with viral load suppression (OR 0.96, 95% CI 0.95–0.97 per unit increase), virological failure (OR 1.05, 95% CI 1.03–1.06) and HIV drug resistance (OR 1.03, 95% CI 1.01–1.05) after adjusting for initial age and CD4 count, previous ART experience, type of regimen and Tuberculosis diagnosis at start of therapy. Similar results were observed in children.

Conclusion

An adherence indicator based on timeliness of clinic attendance predicts strongly both virological response and drug resistance, and could help to timely identify non-adherent patients in settings where viral load monitoring is not available.     

Mapping the distribution of conformational information throughout a protein sequence

The three-dimensional structure of protein is encoded in the sequence, but many amino acid residues carry no essential conformational information, and the identity of those that are structure-determining is elusive. By circular permutation and terminal deletion, we produced and purified 25 Bacillus licheniformis beta-lactamase (ESBL) variants that lack 5-21 contiguous residues each, and collectively have 82% of the sequence and 92% of the non-local atom-atom contacts eliminated. Circular dichroism and size-exclusion chromatography showed that most of the variants form conformationally heterogeneous mixtures, but by measuring catalytic constants, we found that all populate, to a greater or lesser extent, conformations with the essential features of the native fold. This suggests that no segment of the ESBL sequence is essential to the structure as a whole, which is congruent with the notion that local information and modular organization can impart most of the tertiary fold specificity and cooperativity.