Synthesis and biophysical properties of arabinonucleic acids (ANA): circular dichroic spectra, melting temperatures, and ribonuclease H susceptibility of ANA.RNA hybrid duplexes

Arabinonucleic acid (ANA), the 2'-epimer of RNA, was synthesized from arabinonucleoside building blocks by conventional solid-phase phosphoramidite synthesis. In addition, the biochemical and physicochemical properties of ANA strands of mixed base composition were evaluated for the first time. ANA exhibit certain characteristics desirable for use as antisense agents. They form duplexes with complementary RNA, direct RNase H degradation of target RNA molecules, and display resistance to 3'-exonucleases. Since RNA does not elicit RNase H activity, our findings establish that the stereochemistry at C2' (ANA versus RNA) is a key determinant in the activation of the enzyme RNase H. Inversion of stereochemistry at C2' is most likely accompanied by a conformational change in the furanose sugar pucker from C3'-endo (RNA) to C2'-endo ("DNA-like") pucker (ANA) [Noronha and Damha (1998) Nucleic Acids Res. 26, 2665-2671; Venkateswarlu and Ferguson (1999) J. Am. Chem. Soc. 121, 5609-5610]. This produces ANA/RNA hybrids whose CD spectra (i.e., helical conformation) are more similar to the native DNA/RNA substrates than to those of the pure RNA/RNA duplex. These features, combined with the fact that ara-2'OH groups project into the major groove of the helix (where they should not interfere with RNase H binding), help to explain the RNase H activity of ANA/RNA hybrids.     

Impact of growth conditions on the occurrence of<Emphasis Type="Italic">Fusarium</Emphasis> spp. and the mycotoxin content of wheat

In 1997–99 the occurrence ofFusarium spp. on winter wheat and the contamination with mycotoxins was investigated at three locations in the Rhineland, Germany. All cultivation methods investigated had an effect on the level ofFusarium infection, however, rainfall during flowering was the most important factor. The choice of cultivar and soil cultivation proved to be the most promising tools to reduce head scab severity and mycotoxin contamination.     

Recovery of mouse endostatin produced by Pichia pastoris using expanded bed adsorption

Endostatin, a 20 KDa fragment of collagen XVIII, was shown to have an inhibitory effect on angiogenesis and can potentially be used as a tumor growth suppressor. To obtain the amount needed for testing, the protein was successfully cloned and expressed in Pichia pastoris. At the end of the fermentation process, the concentration of the endostatin in the culture was 50 mg per liter, accompanied by 400 gr per liter (wet weight) of biomass. Before the protein can be captured and purified on a packed bed of heparin-Sepharose, the biomass must be removed. Because of the high biomass concentration, conventional biomass removal techniques like centrifugation or filtration are inefficient and cumbersome. Therefore, the expanded-bed adsorption technique was chosen as an alternative approach. An efficient procedure for the initial recovery and purification of the endostatin was developed. The process utilized a cation- exchanger resin instead of a heparin-based affinity resin, because its dynamic capacity was higher, even though it was affected by the high linear flow on the expanded bed. After adjusting the conductivity, pH and biomass concentration, the complete broth was pumped directly on the expanded-bed matrix (Streamline SP XL). Though the yields of protein are similar, the expanded-bed approach is superior to the packed-bed method for several reasons. The expanded-bed process was shorter (only 8 hours compared to 16 hours for the packed bed), it is cheaper, and the product has higher specific activity (29% compared with 18%). Endostatin produced by the expanded-bed adsorption method showed the expected bioactivity and is currently being tested for its potential as a tumor suppressor.     

Generation and characterization of mouse monoclonal antibodies to the myelin-associated glycoprotein (MAG)

A panel of mouse monoclonal antibodies to rat and human myelin-associated glycoprotein (MAG) was developed. Normal mice were unresponsive to rat MAG, and successful immunization with rat MAG was only achieved in autoimmune NZB mice. By contrast, all strains of mice were responsive to human MAG. The monoclonal antibodies developed differ with respect to immunoglobulin type, their specificity for human and/or rat MAG, and their recognition of protein or carbohydrate epitopes in MAG. In general, the antibodies that react with the protein backbone recognize both rat and human MAG, whereas a large number of the monoclonal antibodies recognize a carbohydrate determinant in human MAG that is not in rat MAG. Immunocytochemical staining of adult human spinal cord with the monoclonal antibodies resulted in periaxonal staining of myelin sheaths, similar to that produced by well-defined, rabbit, polyclonal anti-MAG serum. In addition, the antibodies recognizing, carbohydrate determinants in human MAG strongly stained oligodendrocyte cytoplasm. These monoclonal antibodies will be of value for the further chemical and biological characterization of MAG.     

Defined Nutrient Diets Alter Susceptibility to Clostridium difficile Associated Disease in a Murine Model

BackgroundClostridium difficile is a major identifiable and treatable cause of antibiotic-associated diarrhea. Poor nutritional status contributes to mortality through weakened host defenses against various pathogens. The primary goal of this study was to assess the contribution of a reduced protein diet to the outcomes of C. difficile infection in a murine model.MethodsC57BL/6 mice were fed a traditional house chow or a defined diet with either 20% protein or 2% protein and infected with C. difficile strain VPI10463. Animals were monitored for disease severity, clostridial shedding and fecal toxin levels. Select intestinal microbiota were measured in stool and C. difficile growth and toxin production were quantified ex vivo in intestinal contents from untreated or antibiotic-treated mice fed with the different diets.ResultsC. difficile infected mice fed with defined diets, particularly (and unexpectedly) with protein deficient diet, had increased survival, decreased weight loss, and decreased overall disease severity. C. difficile shedding and toxin in the stool of the traditional diet group was increased compared with either defined diet 1 day post infection. Mice fed with traditional diet had an increased intestinal Firmicutes to Bacteroidetes ratio following antibiotic exposure compared with either a 2% or 20% protein defined nutrient diet. Ex vivo inoculation of cecal contents from antibiotic-treated mice showed decreased toxin production and C. difficile growth in both defined diets compared with a traditional diet.ConclusionsLow protein diets, and defined nutrient diets in general, were found to be protective against CDI in mice. Associated diet-induced alterations in intestinal microbiota may influence colonization resistance and clostridial toxin production in a defined nutrient diet compared to a traditional diet, leading to increased survival. However, mechanisms which led to survival differences between 2% and 20% protein defined nutrient diets need to be further elucidated.     

Engineering whole-cell biosensors to evaluate the effect of osmotic conditions on bacteria

Enhanced green fluorescent protein (eGFP) is a variant of wild-type GFP humanized for optimal expression in mammalian cell lines. A computational approach comparing wtGFP and eGFP showed the occurrence of rare proline codons within the eGFP gene that could interfere with and hamper protein production in prokaryotic expression systems. The eGFP gene excised from mammalian plasmid pEGFP N3 was used for construction of two inducible promoter-reporter fusions, T7-eGFP and P_proU-eGFP, through directional cloning. The T7-eGFP fusion confirmed expression of eGFP protein within the bacterial strain, showing a fluorescent green cell pellet and overexpression of the ~29 kDa eGFP protein upon induction with IPTG. The proU operon aids in osmoadaptation by encoding a transport system for uptake of various compatible solutes, including glycine-betaine and proline. Expression of the proU operon is induced upon growth of bacteria in media of elevated osmolarity. When coupled to an eGFP reporter, a time course study using fluorometry demonstrated that induction of P_proU in Escherichia coli occurred rapidly. The P_proU induction and recombinant eGFP production depends on time and concentration of solute (NaCl) in the medium. Cells containing the P_proU-eGFP fusion showed maximum promoter activity at 500 mM concentration of NaCl with a sensitivity of the P_proU promoter being 50 mM. The relative fluorescence reflected the amount of protein synthesized proportional to the activity of induced promoter and effect of NaCl on growth was also taken into consideration. Thus, such environmentally regulated highly sensitive promoters with enhanced reporters could possibly be used as whole-cell biosensors.     

Childhood socioeconomic position and objectively measured physical capability levels in adulthood: a systematic review and meta-analysis

Background

Grip strength, walking speed, chair rising and standing balance time are objective measures of physical capability that characterise current health and predict survival in older populations. Socioeconomic position (SEP) in childhood may influence the peak level of physical capability achieved in early adulthood, thereby affecting levels in later adulthood. We have undertaken a systematic review with meta-analyses to test the hypothesis that adverse childhood SEP is associated with lower levels of objectively measured physical capability in adulthood.

Methods and Findings

Relevant studies published by May 2010 were identified through literature searches using EMBASE and MEDLINE. Unpublished results were obtained from study investigators. Results were provided by all study investigators in a standard format and pooled using random-effects meta-analyses. 19 studies were included in the review. Total sample sizes in meta-analyses ranged from N = 17,215 for chair rise time to N = 1,061,855 for grip strength. Although heterogeneity was detected, there was consistent evidence in age adjusted models that lower childhood SEP was associated with modest reductions in physical capability levels in adulthood: comparing the lowest with the highest childhood SEP there was a reduction in grip strength of 0.13 standard deviations (95% CI: 0.06, 0.21), a reduction in mean walking speed of 0.07 m/s (0.05, 0.10), an increase in mean chair rise time of 6% (4%, 8%) and an odds ratio of an inability to balance for 5s of 1.26 (1.02, 1.55). Adjustment for the potential mediating factors, adult SEP and body size attenuated associations greatly. However, despite this attenuation, for walking speed and chair rise time, there was still evidence of moderate associations.

Conclusions

Policies targeting socioeconomic inequalities in childhood may have additional benefits in promoting the maintenance of independence in later life.     

Citizenship in Question: evidentiary birthright and statelessness

Existing research demonstrates that black population size in a given area correlates with the passage of racially restrictive policies in that area. This paper examines the mechanisms through which minority population size translates into exclusionary policies. It does so by examining a little-known but critical aspect of US civil rights history: the development of policies which allowed white communities to close their public schools entirely rather than desegregate. Using comparative-historical methods to build on existing quantitative studies, this analysis demonstrates that, while black population size does correlate with the passage of restrictive policies, the adoption of school closing policies was primarily a political strategy used to counter rising black political mobilization. That is, whites were not responding to a demographic threat per se or to increasing contact with blacks, as extant work might suggest. Rather, restrictive policies were a response to increasing political activity and mobilization within black communities.     

Scats and den contents as indicators of the diet of stoats (Mustela erminea) in the Tasman Valley,South Canterbury,New Zealand

Stoats are significant predators of native fauna in New Zealand. They occur in many habitat types and consume a wide range of prey. The diet of stoats in the Tasman River, South Canterbury, was studied by analysis of scats and den contents. Analysis of 206 scats showed that stoats ate mainly lagomorphs, birds and invertebrates. Minor components included mice, lizards, fish and hedgehogs. Stoats ate more birds in spring than in autumn, and female stoats ate more invertebrates than did males. The contents of 219 dens collected in the same area at the same time provided further information. Birds and lagomorphs occurred at high frequency in dens, and other components were minor. Remains in dens were larger than in scats and allowed identification of many more prey items to species level. Den contents revealed a potentially substantial impact of stoats on threatened shorebirds locally; this impact was not detected by analysis of scats.