Arenesulfonylethoxycarbonyl- a Set of Amino Protecting Groups for DNA and RNA Synthesis

Abstract

Phenyl-, 4-chlorophenyl- and 4-nitrophenylsufonylethoxycarbonyl groups have been reported for the first time as the exocyclic amino protecting groups in nucleoside chemistry. They are all stable under the standard conditions of manipulations in phosphotriester and phosphiteamidite chemistry, they are removable both under the alkaline hydrolytic conditions and also under the influence of non-nucleophilic tertiary bases. N³-Phenyl- and 4-toluenesulfonylethoxycarbonyl derivatives of uridine have been also prepared and characterized by ¹⁵N-NMR spectroscopy, their stabilities under different conditions have been tested.     

On the oxygen-sensitivity of various tetrazolium salts

Summary 1. Eight different tetrazolium salts have been chemically reduced with NADPH and PMS¹ under oxygenated and oxygen-free conditions. 2. PMS has been shown to be able to remove all of the hydrogen from NADPH very rapidly, and to transfer all of this hydrogen onto tetrazolium salts, under suitable atmospheric conditions. 3. MTT, INT, TNBT, and NBT¹ produced the same amount of formazan under both conditions; NT BT, TV, TT¹ produced formazan under oxygen-free conditions, but produced no formazan under oxygenated conditions. 4. These results are explained on the basis of competition for the NADP Hhydrogen between oxygen and the four tetrazolium salts NT, BT, TV and TT.I should like to thank The Arthritis and Rheumatism Council for financial support.     

Evidence for A Ca2+ gradient across the plasma membrane of wheat protoplasts

Fluxes of Ca²⁺ across the plasma membrane of isolated wheat protoplasts have been measured both as net accumulation and as uptake under steady-state conditions. The ATPase inhibitors, orthovanadate and diethylstibesterol, and the divalent cation ionophore, A23187, were all found to enhance net Ca²⁺ accumulation by protoplasts. The uptake of Ca²⁺ under steady-state conditions was also stimulated by A23187 but relatively unaffected by a range of plant hormones or by red or far red light. Light treatments were compared to dark controls with protoplasts isolated from etiolated wheat.The results suggest that plant cells maintain a Ca²⁺ gradient across their plasma membrane but it appears not to be under phytochrome control.     

A Multifaceted Independent Performance Analysis of Facial Subspace Recognition Algorithms

Face recognition has emerged as the fastest growing biometric technology and has expanded a lot in the last few years. Many new algorithms and commercial systems have been proposed and developed. Most of them use Principal Component Analysis (PCA) as a base for their techniques. Different and even conflicting results have been reported by researchers comparing these algorithms. The purpose of this study is to have an independent comparative analysis considering both performance and computational complexity of six appearance based face recognition algorithms namely PCA, 2DPCA, A2DPCA, (2D)²PCA, LPP and 2DLPP under equal working conditions. This study was motivated due to the lack of unbiased comprehensive comparative analysis of some recent subspace methods with diverse distance metric combinations. For comparison with other studies, FERET, ORL and YALE databases have been used with evaluation criteria as of FERET evaluations which closely simulate real life scenarios. A comparison of results with previous studies is performed and anomalies are reported. An important contribution of this study is that it presents the suitable performance conditions for each of the algorithms under consideration.     

Continuous-flow NMR culture system for mammalian cells

A continuous-flow NMR culture system for mammalian cells has been developed on which ³¹P-NMR experiments under complete and strictly physiologic conditions have been performed. Observations on the response of the cellular metabolism to stresses such as starvation, low temperature and changes in environmental pH monitored by ³¹P-NMR are reported. The response of the intracellular pH relative to the external pH of the growth medium is studied. We find that under the experimental conditions used there exists a ΔpH varying between less than 0.2 and more than 0.6 pH units. These results are compatible with those obtained using other techniques.     

Some effect of sodium application on the yield and chemical composition of pasture grown under differing conditions of potassium and moisture supply

The effects of Na fertiliser (supplied as an NPK-Na compound) on herbage yield and composition were examined at two different sites to see if pasture responses to Na were affected by differences in K and moisture availability. At one site, pasture was grown under conditions of moisture stress and limited K availability, whereas at the other site the pasture was grown under comparatively non-stress conditions.The results were interesting in that Na fertilisation appeared to be detrimental to pasture yield and quality under conditions of moisture stress and suboptimal K supply, whereas under the non-stress conditions it actually increased herbage N offtake, marginally improved the nutritional quality of the pasture and produced appreciable (albeit non-significant) increases in DM yield. It was suggested that an important effect of Na on grass production may have been its ability to enhance the rate of NO₃ ^– uptake by plants, thereby minimising NO₃ ^–-N losses from the soil-plant system by denitrification. However, because the amount of N fertiliser used in the experiments (i.e. 390 kg N ha^–1 yr^–1) was close to that normally required for maximum yield production (N max) under Northern Ireland conditions, the scope for yield increases in response to Na-elicited improvements in N offtake were thought to have been very limited at both experimental sites.On the basis of results from both the present study and previous field trials, a unifying theory is presented to explain the differential effects of Na on NO₃ ^– uptake and herbage growth under different sets of circumstances.     

Insight into a Random Coil Conformation and an Isolated Helix: Structural and Dynamical Characterisation of the C-Helix Peptide from Hen Lysozyme

A 17 residue peptide corresponding to the C-helix of hen lysozyme (residues 86 to 102) has been investigated in detail to assess the factors that determine its conformation in both aqueous and trifluoroethanol (TFE) solutions. A thorough characterisation of the peptide by CD and NMR techniques under both conditions has been performed including the determination of complete NMR proton sequential assignments, and measurement of NOE effects,³J_HNαcoupling constants, temperature coefficients and residue-specific hydrogen-exchange rates. In water, the peptide adopts a largely unstructured conformation and NMR data, particularly coupling constants and chemical shift deviations, have been shown to agree closely with predictions from a model for a random coil based on the φ,ψ distributions in a protein database. This indicates that under these conditions the intrinsic conformational preferences of the individual amino acid residues are the dominating factors that determine the population of conformers adopted. With increasing concentrations of TFE a cooperative transition to an extensively helical conformation occurs and the resultant changes in CαH chemical shifts have been shown to correlate with the changes in φ,ψ populations. Using NOE and coupling constant data for this state, an ensemble of structures has been calculated and provides a model for a helix in the absence of tertiary interactions. In this model fluctuations, which increase in amplitude towards the termini, occur about the average helical φ,ψ angles and are responsible for increasing the values of³J_HNαcoupling constants above those anticipated for a static helix. The residue-specific rates of hydrogen exchange for the peptide in 50% TFE-d₃are consistent with such a model, the maximum protection from exchange being observed for residues in the centre of the helix.     

Carbon balance during CAM: an assessment of respiratory CO2 recycling in the epiphytic bromeliads Aechmea nudicaulis and Aechmea fendleri

Abstract The regulation of crassulacean acid metabolism (CAM) under controlled environmental conditions has been investigated for two tropical epiphytes, relating plant water and carbon balance to growth form and habitat preference under natural conditions. Aechmea fendleri is restricted to wet, upper montane regions of Trinidad, while A. nudicaulis has a wider distribution extending into more arid regions of the island. Morphological characteristics of these plants are related to habitat preference in terms of leaf succulence (0.44 and 0.94 kg m^?2 for the two species respectively) and a distinct layer of water storage parenchyma in A. nudicaulis In contrast, the thinner leaves of A. fendleri contain little water-storage parenchyma and less chlorenchyma per unit area, but the plants have a more open leaf rosette. The two species differ in expression of CAM, since the proportion of respiratory CO₂ recycled as part of CAM had been found to be much lower in A. fendleri This study compared the efficiency of water use and role of respiratory CO₂ recycling under two PAR regimes (300 and 120 μnol m^?2 s^?1) and three night temperatures (12, 18 and 25 °C). Dark CO₂ uptake rates for both species were comparable to plants in the field (maximum of 2.3 ± 0.2 μmol m^?2s^?1± SD, n= 3). Total net CO₂ uptake at night increased on leaf area basis with temperature for both species under higher PAR, although under the low PAR regime CO₂ uptake was maximal at 18 °C. Water-use efficiency (WUE) increased at 18 °C and 25 °C during dark CO₂ uptake (Phase I) and also during late afternoon photosynthesis (Phase IV) in both species. For A. fendleri, dawn to dusk changes in titrable acidity (ΔH ⁺) were similar under high and low PAR, although ΔH⁺ was correlated to night temperature and PAR in A. nudicaulis. The proportion of ΔH⁺ derived from respiratory CO₂ also varied with experimental conditions. Thus percentage recycling was lower in A. fendleri under high PAR (0–10%), but was only reduced at 18 °C under low PAR. Recycling by A. nudicaulis ranged from 32–42% under high PAR, but was also reduced to 6% under low PAR at 18 °C; at 12 °C and 25 °C, recycling was 37% and 52% respectively. Previous studies have suggested a relationship between the proportion of recycling and degree of water stress. This study indicated that CAM as a CO₂ concentrating mechanism regulates both water-use efficiency and plant carbon balance in these epiphytes, in response to PAR and night temperature. However, the precise relationship between respiratory processes and the balance between external and internal sources of CO₂ is as yet unresolved.     

Optimization of red light-induced elongation in Avena coleoptile sections and properties of the phytochrome-mediated growth response*

Abstract Optimal conditions for studying the elongation response to a 1 mmol m^?2, 2-min pulse of red light in subapical coleoptile sections from dark-grown oat (Avena sativa L. ev. Lodi) seedlings have been determined. A technique for obtaining standard-length coleoptile sections without exposing either seedlings or sections to any light has been developed, and is described. The optimal conditions found were: sampling time, 12 h after irradiation; buffer conditions, 5 mol m^?3 potassium phosphate with 5% (w/v) sucrose (pH 5.9). The optima were determined by obtaining the time course for light-induced growth under various conditions. The red light-induced growth response is linear until 12 h after irradiation, when it undergoes an interruption. Optimal incubation conditions were determined by varying the buffer contents systematically and measuring the responses at the optimal lime determined. The results indicate a distinct difference between auxin-induced and light-induced growth responses. Even with variations of basal growth rate and several incubation conditions, the red light-induced elongation appears to be of a constant magnitude, to persist for a constant time period. and to exhibit a constant lag period between irradiation and the onset of response. The use of sections that were produced and handled in complete darkness yielded an unusual response to fusicoccin. A linear, high growth rate in response to I mmol m^?3 FC was observed for more than 12 h, both in the irradiated sections and in the dark controls.     

Remodelling of arabinoxylan in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) endosperm cell walls during grain filling

Previous studies using spectroscopic imaging have allowed the spatial distribution of structural components in wheat endosperm cell walls to be determined. FT-IR microspectroscopy showed differing changes in arabinoxylan (AX) structure, during grain development under cool/wet and hot/dry growing conditions, for differing cultivars (Toole et al. in Planta 225:1393–1403, 2007). These studies have been extended using Raman microspectroscopy, providing more details of the impact of environment on the polysaccharide and phenolic components of the cell walls. NMR studies provide complementary information on the types and levels of AX branching both early in development and at maturity. Raman microspectroscopy has allowed the arabinose:xylose (A/X) ratio in the cell wall AX to be determined, and the addition of ferulic acid and related phenolic acids to be followed. The changes in the A/X ratio during grain development were affected by the environmental conditions, with the A/X ratio generally being slightly lower for samples grown under cool/wet conditions than for those from hot/dry conditions. The degree of esterification of the endosperm cell walls with ferulic acid was also affected by the environment, being lower under hot/dry conditions. The results support earlier suggestions that AX is either delivered to the cell wall in a highly substituted form and is remodelled through the action of arabinoxylan arabinofuranohydrolases or arabinofuranosidases, or that low level substituted AX are incorporated into the wall late in cell wall development, reducing the average degree of substitution, and that the rate of this remodelling is influenced by the environment. ¹H NMR provided a unique insight into the chemical structure of intact wheat endosperm cell walls, providing qualitative information on the proportions of mono- and disubstituted AX and the levels of branching of adjacent units. The A/X ratio did not change greatly with either the development stage or the growth conditions, but the ratio of mono- to disubstituted Xylp residues increased markedly (by about fourfold) in the more mature samples, confirming the changes in branching levels determined using FT-IR. To the best of our knowledge, this is the first time that intact endosperm cell walls have been studied by ¹H NMR.     

Taurine release in mouse brain stem slices under cell-damaging conditions

Summary. Taurine has been thought to be essential for the development and survival of neural cells and to protect them under cell-damaging conditions. In the brain stem taurine regulates many vital functions, including cardiovascular control and arterial blood pressure. We have recently characterized the release of taurine in the adult and developing brain stem under normal conditions. Now we studied the properties of preloaded [³H]taurine release under various cell-damaging conditions (hypoxia, hypoglycemia, ischemia, the presence of metabolic poisons and free radicals) in slices prepared from the mouse brain stem from developing (7-day-old) and young adult (3-month-old) mice, using a superfusion system. Taurine release was greatly enhanced under these cell-damaging conditions, the only exception being the presence of free radicals in both age groups. The ischemia-induced release was characterized to consist of both Ca²⁺-dependent and -independent components. Moreover, the release was mediated by Na⁺-, Cl⁻-dependent transporters operating outwards, particularly in the immature brain stem. Cl⁻ channel antagonists reduced the release at both ages, indicating that a part of the release occurs through ion channels, and protein kinase C appeared to be involved. The release was also modulated by cyclic GMP second messenger systems, since inhibitors of soluble guanylyl cyclase and phosphodiesterases suppressed ischemic taurine release. The inhibition of phospholipases also reduced taurine release at both ages. This ischemia-induced taurine release could constitute an important mechanism against excitotoxicity, protecting the brain stem under cell-damaging conditions.     

Monitoring Protein Adsorption with Solid-state Nanopores

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ^1-6, the unfolding of proteins ⁷, and binding affinity of different ligands ⁸. By coupling these nanopores to the resistive-pulse technique ^9-12, such measurements can be done under a wide variety of conditions and without the need for labeling ³. In the resistive-pulse technique, an ionic salt solution is introduced on both sides of the nanopore. Therefore, ions are driven from one side of the chamber to the other by an applied transmembrane potential, resulting in a steady current. The partitioning of an analyte into the nanopore causes a well-defined deflection in this current, which can be analyzed to extract single-molecule information. Using this technique, the adsorption of single proteins to the nanopore walls can be monitored under a wide range of conditions ¹³. Protein adsorption is growing in importance, because as microfluidic devices shrink in size, the interaction of these systems with single proteins becomes a concern. This protocol describes a rapid assay for protein binding to nitride films, which can readily be extended to other thin films amenable to nanopore drilling, or to functionalized nitride surfaces. A variety of proteins may be explored under a wide range of solutions and denaturing conditions. Additionally, this protocol may be used to explore more basic problems using nanopore spectroscopy.     

Influence of N and K fertilization and growth temperature on 13C/12C ratios of timothy (Phleum pratense L.)

Summary Measurements have been reported of carbon isotope ratios of timothy grown at different temperatures and with varying nitrogen and potassium supplies. Both total plant tissue and extracted plant tissue have been analyzed. The ¹³C/¹²C ratios were found to vary both with temperature and with nutrient level; the highest values of ¹³C were found under the most optimum growth conditions.     

EXPRESSION OF GENETIC VARIATION IN BODY SIZE OF THE COLLARED FLYCATCHER UNDER DIFFERENT ENVIRONMENTAL CONDITIONS

Heritability of body size in two experimentally created environments, representing good and poor feeding conditions, respectively, was estimated using cross-fostered collared flycatcher Ficedula albicollis nestlings. Young raised under poor feeding conditions attained smaller body size (tarsus length) than their full-sibs raised under good feeding conditions. Parent-offspring regressions revealed lower heritability (h²) of body size under poor than under good feeding conditions. Hence, as the same set of parents were used in the estimation of h² in both environments, this suggests environment-dependent change in additive genetic component of variance (V_A), or that the genetic correlation between parental and poor offspring environment was less than that between parental and good offspring environment. However, full-sib analyses failed to find evidence for genotype-environment interactions, although the power of these tests might have been low. Full-sib heritabilities in both environments tended to be higher than estimates from parent-offspring regressions, indicating that prehatching or early posthatching common environment/maternal effects might have inflated full-sib estimates of V_A. The effect of sibling competition on estimates of V_A was probably small as the nestling size-hierarchy at day 2 posthatch was not generally correlated with size-hierarchy at fledging. Furthermore, there was no correlation between maternal body condition during the incubation and final size of offspring, indicating that direct maternal effects related to nutritional status were small. A review of earlier quantitative genetic studies of body size variation in birds revealed that in eight of nine cases, heritability of body size was lower in poor than in good environmental conditions. The main implication of this relationship will be a decreased evolutionary response to selection under poor environmental conditions. On the other hand, this will retard the loss of genetic variation by reducing the accuracy of selection and might help explain the moderate to high heritabilities of body-size traits under good environmental conditions.     

Identification and partial characterization of multiple discrete polyadenylic acid containing RNA components coded for by HeLa cell mitochondrial DNA

Poly(A)-containing RNA isolated from the components of a HeLa cell mitochondrial lysate which sediment in the polysome region of a sucrose gradient have been analyzed for the presence of discrete species. Eight distinct components have been identified by polyacrylamide gel electrophoresis after formaldehyde treatment. These components, which are highly reproducible in their occurrence and relative amounts under widely varying conditions of isolation, have been characterized as to their sedimentation behavior under denaturing conditions, poly(A) content and homology to separated strands of mitochondrial DNA.One of the discrete components was previously shown to have a sedimentation coefficient of about 7 S in the native state and a molecular weight of about 9.0 × 10⁴, as estimated from its sedimentation rate in formaldehyde. The molecular weights of the other seven components, as derived from sedimentation data, range between 2.6 and 5.3 × 10⁵.The 7 S RNA is complementary to the light mitochondrial DNA strand, while the other seven components are complementary to the heavy strand. Together with the two mitochondrial rRNA species and with mitochondrial 4 S RNA, the eight poly(A)-containing RNA components, if distinct in sequence, would account for about 70% of the single-strand informational content of HeLa mitochondrial DNA.     

Emerging roles for sodium dependent amino acid transport in mesenchymal cells

Summary The functional aspects of sodium dependent amino acid transport in mesenchymal cells are the subject of this contribution. In a survey of the cross-talk existing among the various transport mechanisms, particular attention is devoted to the role played by substrates shared by several transport systems, such as L-glutamine. Intracellular levels of glutamine are determined by the activity of System A, the main transducer of ion gradients built on by Na,K-ATPase into neutral amino acid gradients. Changes in the activity of the System are employed to regulate intracellular amino acid pool and, hence, cell volume. System A activity has been found increased in hypertonically shrunken cells and in proliferating cells. Under both these conditions cells have to increase their volume; therefore, System A can be employed as a convenient mechanism to increase cell volume both under hypertonic and isotonic conditions. Although less well characterized, the uptake of anionic amino acids performed by System X^– _AG may be involved in the maintenance of intracellular amino acid pool under conditions of limited availability of neutral amino acids substrates of System A.     

Fluid compartmentation and electrolytes of cat cerebral cotex in vitro. 3. Ontogenetic and comparative aspects

Studies on swelling and fluid compartmentation have been carried out in vitro on incubated slices of cerebral cortex from kittens 1.5-120 days post-natal age and on incubated sections of corpus callosum and slices of liver and kidney cortex from adult cats. The findings have been compared with analogous data for incubated slices of adult cat cerebral cortex, studied under identical conditions (Bourke and Tower , 1966a, b), in order to identify the probable morphological correlates of fluid and electrolyte distribution. Incubated cortical slices from neonatal (1.5-4-day-old) kittens exhibit none of the relevant characteristics of slices from adult cerebral cortex. By 1 month post-natal age, K⁺-dependent swelling of slices becomes demonstrable, and the K⁺ and Na⁺ contents of slices approximate adult levels. Both these developments coincide with the morphological and physiological maturation of cortical neurons. At 3 months post-natal age, slice swelling accessible to C1^? but not to sucrose becomes observable and the dependence of sucrose space size on time, during incubation, of solute addition becomes demonstrable. Both these developments follow completion of axonal myelination in the cortex but coincide with the period of cortical glial cell proliferation. Incubated sections of corpus callosum from adult cats exhibit none of the relevant characteristics observed for cortical slices under identical conditions. Tissue swelling is minimal and uninfluenced by K⁺ concentrations of incubation media. Tissue fluid spaces accessible to sucrose are approximately twice the size of spaces accessible to inulin. In general, qualitatively similar results have been obtained for incubated slices of cat liver or kidney cortex or for incubated sections of rat diaphragm under the same conditions. A behaviour for glial cells (? astrocytes) in cerebral cortex under such in vitro conditions distinctly different from behaviour of subcortical glial cells is suggested.     

Microaerophilic degradation of hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (RDX) by three Rhodococcus strains

Aim: The goal of this study was to compare the degradation of hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (RDX) by three Rhodococcus strains under anaerobic, microaerophilic (<0·04 mg l^?1 dissolved oxygen) and aerobic (dissolved oxygen (DO) maintained at 8 mg l^?1) conditions. Methods and Results: Three Rhodococcus strains were incubated with no, low and ambient concentrations of oxygen in minimal media with succinate as the carbon source and RDX as the sole nitrogen source. RDX and RDX metabolite concentrations were measured over time. Under microaerophilic conditions, the bacteria degraded RDX, albeit about 60‐fold slower than under fully aerobic conditions. Only the breakdown product, 4‐nitro‐2,4‐diazabutanal (NDAB) accumulated to measurable concentrations under microaerophilic conditions. RDX degraded quickly under both aerated and static aerobic conditions (DO allowed to drop below 1 mg l^?1) with the accumulation of both NDAB and methylenedinitramine (MEDINA). No RDX degradation was observed under strict anaerobic conditions. Conclusions: The Rhodococcus strains did not degrade RDX under strict anaerobic conditions, while slow degradation was observed under microaerophilic conditions. The RDX metabolite NDAB was detected under both microaerophilic and aerobic conditions, while MEDINA was detected only under aerobic conditions. Impact and Significance of the Study: This work confirmed the production of MEDINA under aerobic conditions, which has not been previously associated with aerobic RDX degradation by these organisms. More importantly, it demonstrated that aerobic rhodococci are able to degrade RDX under a broader range of oxygen concentrations than previously reported.     

Mapping the enzymatic active site of Pseudomonas aeruginosa exotoxin A

Pseudomonas aeruginosa exotoxin A is representative of a class of enzymes, the monoADP-ribosyl, which catalyze the covalent transfer of an ADP-ribose moiety of NAD⁺ to a target substrate. Availability of the three-dimensional structure of exotoxin A provides the opportunity for mapping substrate binding sites and suggesting which amino acid residues may be involved in catalysis. Data from several sources have been combined to develop a proposal for the NAD⁺ binding site of exotoxin A: the binding of NAD⁺ fragments adenosine, AMP, and ADP have been delineated crystallographically to 6.0, 6.0, and 2.7 Å, respectively; significant sequence homology spanning 60 residues has been found between exotoxin A and diphtheria toxin, which has the identical enzymatic activity; iodination of exotoxin A, under conditions in which only tyrosine 481 is iodinated in the enzymatic domain, abolishes ADP-ribosyl transferase activity.