Root length and diameter measurement using NIH Image: application of the line-intercept principle for diameter estimation

The objective of this study was to develop an image analysis algorithm for estimating the length versus diameter distribution of washed root samples. Image analysis was performed using a Macintosh computer and the public domain NIH Image program. After an appropriate binary image of roots was obtained, the image was processed to get the thinned image to calculate the length of the roots. The edge pixel of the binary image was then deleted and root length was calculated again. This `edge deletion–length calculation' cycle was repeated until no root pixel was left in the image. Repeated edge deletion removed one pixel layer from around the periphery of root objects in each iteration. The number of edge deletions, which is equivalent to the intercept length, can be used to estimate the root diameter. We used the vertical or horizontal intercept length, whichever was shorter. The accuracy of diameter estimation due to orientation of objects varied from 89.1 to 126.0%. Branching root systems consist of several orders of laterals, and as the root branches to a higher order, the diameter of the roots becomes smaller. Therefore, edge deletions eliminate sequentially from the highest order roots, which have the smallest diameter, to the lowest order roots, which have the widest diameter. Thus, the length and diameter of each root order can be calculated by the proposed method. For verification, images of copper wire of 0.23, 0.50, and 1.0 mm diameter were analyzed. The results showed reasonable agreement with the expected distribution of length versus diameter for randomly oriented objects, and consequently the wire length of each diameter could be estimated. The proposed method was tested for primary and secondary roots of water-cultured rice (Oryza sativa L.), and it was proven that the method can provide accurate length and diameter measurements for each root order.     

Accurate root length and diameter measurement using NIH Image: use of Pythagorean distance for diameter estimation

This report describes an image analysis algorithm to estimate the length versus diameter of washed root samples accurately. Image analysis was performed using a Macintosh computer and the public domain NIH Image program. The binary image of the roots was processed to get the thinned image to calculate the length of the roots. The pixels of the root in a binary image were then stripped off from around the periphery based on the pixel's Pythagorean distance from the nearest background pixel. The length of the remaining root in each stripping off process was calculated after the image was thinned. Images (300 dpi) of copper wire of 0.23, 0.5, 1.0 mm diameter were analyzed for verification of the usefulness of the procedure. The results showed that more than 93% of the wires in each diameter wire were calculated to be in diameter classes including the true diameter and its adjoining classes: 93.6% of the wires of 0.23 mm diameter appeared in the 0.098–0.38 mm diameter classes, 96.19% of the wires of 0.5 mm diameter appeared in the 0.38–0.61 mm diameter classes, and 96.17% of the wires of 1 mm diameter appeared in the 0.85–1.08 mm diameter classes. The proposed method was tested for primary and secondary roots of water-cultured rice (Oryza sativa L.) and it was proven that the method could provide accurate length and diameter measurements for each root order. In addition, it was found that the method could provide the lengths of the thick primary, thin primary, and secondary roots. The effectiveness of applying sharpening for the grayscale image before making the binary image is also discussed.     

Analysis of root images from auger sampling with a fast procedure: a case of application to sugar beet

Manual line-intersect methods for estimating root length are being progressively replaced by faster and more accurate image analysis procedures. These methods even allow the estimation of some more root parameters (e.g., diameter), but still require preliminary labour-intensive operations. Through a task-specific macro function written in a general-purpose image analysis programme (KS 300 – Zeiss), the processing time of root images was greatly reduced with respect to skeletonisation methods by using a high-precision algorithm (Fibrelength). This has been previously proposed by other authors, and estimates length as a function of perimeter and area of the digital image of roots. One-bit binary images were acquired, aiming at large savings in computer memory, and automatic discrimination of roots against extraneous objects based on their elongation index (perimeter²/area), was performed successfully. Of four tested spatial resolutions (2.9, 5.9, 8.8, 11.8 pixel mm^–1), in clean samples good accuracy in root length estimation was achieved at 11.8 pixel mm^–1, up to a root density of 5 cm cm^–2 on the scanner bed. This resolution is theoretically suitable for representing roots at least 85 m wide. When dealing with uncleaned samples, a thick layer of water was useful in speeding up spreading of roots on the scanner bed and avoiding underestimation of their length due to overlaps with organic debris. A set of fibrous root samples of sugar beet (Beta vulgaris var. saccharifera L.) collected at harvest over two years at Legnaro (NE Italy) was analysed by applying the above procedure. Fertilisation with 100 kg ha^–1 of nitrogen led to higher RLD (root length density in soil) in shallow layers with respect to unfertilised controls, whereas thicker roots were found deeper than 80 cm of soil without nitrogen.     

Issues in using the WinRHIZO system to determine physical characteristics of plant fine roots

Image analysis systems have facilitated rapid measurements of fine root length (RL), diameter (RD), volume (RV), etc. The WinRHIZO system is unlike other image analysis systems in that it can detect, and make corrections for, areas of root overlap. It is designed to be capable of using both Regent’s non-statistical method (WinRHIZO method) and Tennant’s statistical method (line-intersect method), and can simultaneously output the root measurements by both methods when they are chosen at the same time. This study tested: (1), the efficacy of the overlap correction function in the WinRHIZO system; and (2), the consistency of fine root measurements between the WinRHIZO and Tennant methods with two sets of root measurement data from winter wheat (Triticum aestivium L.). The results showed that there were significant differences in RL, RD and RV between small root samples with and without stumps. The impact of root stumps outweighed the overlap correction efficacy in WinRHIZO. The values from the Tennant method are significantly different from those using the WinRHIZO method, although both results are statistically closely correlated. This indicated how critical it was to use without-stump root samples when using image analysis systems to measure RL, RD, RV, etc., and to keep in mind that a significant difference in root measurements may be methodologically related when comparing the results of various experiments from these two methods. Our research results bear important implications for the study of root ecology.     

A simple apparatus for generating stretched polyacrylamide gels, yielding uniform alignment of proteins and detergent micelles*

Compressed and stretched polyacrylamide hydrogels previously have been shown to offer a robust method for aligning proteins. A simple, funnel-like apparatus is described for generating uniformly stretched hydrogels. For prolate-shaped proteins, gels stretched in the direction of the magnetic field yield two-fold larger alignment than gels compressed to the same aspect ratio in this direction. Empirically, protein alignment is found to be proportional to (c–2.3)² [(d_o/d_N)³-1], where d_o and d_N are the diameters of the cylindrical gels before and after stretching, respectively, and c is the polyacrylamide weight fraction in percent. Low gel densities, in the 4–7% range, are found to have minimal effects on macromolecular rotational correlation times, _c, and no effect of the compression ratio on _c could be discerned over the range studied (d_o/d_N le1.4). Application is demonstrated for a sample containing the first Ig-binding domain of protein G, and for a detergent-solubilized peptide.     

Purification and some Properties of β-Xylosidase from Trichoderma viride

β-Xylosidase was purified 25 fold from a culture filtrate by ammonium sulfate fractionation, DEAE-Sephadex chromatography, column electrophoresis, gel filtration on Biogel P-100, and isoelectric focusing. The purified β-xylosidase was found to be homogeneous on SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis and on disc electrophoresis. A molecular weight of 101,000 was estimated by chromatography on Sephadex G-200, and 102,000 was obtained by SDS polyacrylamide gel electrophoresis. The purified p-xylosidase had an isoelectric point at pH 4.45, and contained 4.5% carbohydrate residue. The optimum activity for the enzyme was found to be at pH 4.5 and 55°C. The enzyme activity was inhibited by Hg^{2 +}, and N-bromosuccinimide at a concentration of 1 x 10^?3 m. The purified enzyme hydrolyzed phenyl β-d-xyloside (k_o—13.0 sec”¹), p-nitrophenyl β-d-xyloside (k_o=2l.3 sec^?1), o-nitrophenyl β-d-xyloside (k_o = 22.2 sec^?1), o-chlorophenyl β-d-xyloside (k_o = 20.0 sec^?1), p-methylphenyl β-d-xyloside (k_o~9.0 sec^?1), o-methylphenyl β-d-xyloside (k_o= 10.7 sec^?1), p-methoxyphenyl β-d-xyloside (k_o=10.3 sec^?1), o-methoxyphenyl β-d-xyloside (&;_o=10.9 sec^?1), xylobiose (k_o = 36A sec^?1), xylotriose (k_o = 34.5 sec^?1), xylotetraose (k_o~HA sec^?1), and xylopentaose (k_o= 13.0 sec^?1). On enzymic hydrolysis of phenyl β-d-xyloside, the reaction product was found to be β-d-xylose with retention of configuration. The purified p-xylosidase was practically free of α-xylosidase and β-glucosidase activities.     

What limits nitrate uptake from soil?

Abstract. An accepted view, that unless nitrate concentrations in the soil solution are very low (e.g. below 0.1–0.2 mol m^?3) the growth of high-yielding crops is not limited by the availability of nitrogen, is challenged. Conventional analyses of nutrient supply and demand, based on calculations of apparent inflow rates (uptake rates per unit total root length) are invalid. Apparent inflow rates are inversely proportional to root length. The convention of using total root length grossly overestimates the fraction of the root system active in nutrient uptake. Consequently, inflow rates based on total root lengths underestimate the true values, indicating unrealistically low nutrient concentration differentials between bulk soil and root surfaces required to drive uptake. An alternative method of analysis is suggested. This is based on total nutrient uptake rather than on inflow rate. Measurements of the former do not depend on estimates of active root length and can be made directly and reliably. The method was applied to data obtained from a pot experiment using spring wheat (Triticum aestivum L., cv. Wembley) grown in soil without nitrogen fertilizer (N₀) or with nitrogen fertilizer equivalent to 200kg N ha^?1 (N⁺). Soil nitrate concentrations calculated using the conventional method based on total root length, suggested that any increases in concentration above those measured in the N⁰ treatment should not have resulted in increased uptake and growth. However, the N⁺ plants were always bigger than those in the No treatment, refuting this suggestion. Theoretical uptakes of nitrogen (calculated initially on the basis of a fully active root system) were adjusted, by reducing the effective root length incrementally, until the theoretical uptake matched the measured net uptake of nitrogen. The mean fractions of the root systems likely to have been involved in nitrate uptake were 11% and 3.5% of the total lengths of root in the N⁰ and N⁺ treatments, respectively.     

Purification and Some Properties of β-Xylosidase from Emericella nidulans

β-Xylosidase was purified 662 fold from a culture filtrate by ammonium sulfate fractionation, gel filtration on Biogel P-100, DEAE-Sephadex chromatography, and gel filtration on Sephadex G-200. With isoelectric focusing, the purified β-xylosidase found to be homogeneous on SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis. The molecular weight was estimated by gel filtration to be 240,000, and 116,000 by SDS polyacrylamide gel electrophoresis. The purified β-xylosidase had an isoelectric point at pH 3.25, and contained 4% carbohydrate residue. The optimum pH was found to be in the range of 4.5 ~ 5, and the optimum temperature was 55°C. The enzyme activity was inhibited by Hg^{2 +}, SDS, and N-bromosuccinimide at a concentration of 1 × 10^?3 m, and also p-chloromercuribenzoate at a concentration of 1 × 10^?4m. The purified enzyme hydrolyzed phenyl β-d-xyloside (k_o = 302.6 sec^?1),β-nitrophenyl β-d-xyloside (k_o = 438.9 sec^?1), o-nitrophenyl β-d-xyloside (k_o = 431.0 sec^?1), p-chlorophenyl β-d-xyloside (k_o = 207.9 sec^?1), o-chlorophenyl β-d-xyloside (k_o = 211.8 sec^?1), β-methylphenyl β-d-xyloside k_o = 96.5 sec^?1), o-methylphenyl β-d-xyloside (k_o = 83.1 sec^?1), p-methoxyphenyl β-d-xyloside (k_o = 99.3 sec^?1), o-methoxyphenyl β-d-xyloside (k_o= 100.0 sec^?1), xylobiose (k_o = 992A sec^?1), xylotriose (k_o = 1321.9 sec^?1), xylotetraose (k_o = 7S9.1 sec^?1) and xylopentaose (k_o = 508.0 sec^?1). On enzymic hydrolysis of phenyl β-d-xyloside, the reaction product was found to be β-d-xylose with retention of the configuration. The purified β-xylosidase was practically free of a-xylosidase and β-glucosidase activities.     

Cytokinin inhibition of Arabidopsis root growth: An examination of genotype, cytokinin activity, and N6-benzyladenine metabolism

The effects of cytokinins on the in vitro growth of the roots of Arabidopsis thaliana seedlings were examined. Root growth was inhibited in a manner dependent upon the type of cytokinin compound, the cytokinin concentration, the Arabidopsis genotype, and the duration of exposure to cytokinin. For the cytokinins N ⁶-benzyladenine (BA), isopentenyl adenine (iP), or dihydrozeatin (DHZ), the concentration required for 50% root growth inhibition differed for each cytokinin and in each of three Arabidopsis genotypes tested. iP was the most active cytokinin in inhibiting the root growth of the Ler-0 genotype, whereas iP and BA had equal activity when tested with the Col-2 and Columbia genotypes. DHZ had the lowest activity of the three cytokinins tested in all three genotypes. A brief 1-day exposure of seeds to a root-inhibiting concentration of BA increased root growth compared with seedlings grown without BA; exposure to BA for 3–6 days inhibited root growth. BA metabolism was evaluated after 6 h and 1, 3, and 6 days in Columbia seedlings. BA, N ⁶-benzyladenosine (BAR), and N ⁶-benzyladenosine-5-monophosphate (BAMP) decreased with time, whereas N ⁶-benzyladenine-7--d-glucopyranoside (BA-7-G) and N ⁶-benzyladenine-9--d-glucopyranoside (BA-9-G) accumulated in the growing seedlings. Seven aromatic cytokinins were compared at 5 m for their effect on Col-3 root growth. BA, BAR, N ⁶-(m-hydroxybenzylamino)adenine, and N ⁶-(o-hydroxybenzylamino)adenine were highly effective in inhibiting root growth, whereas N ⁶-(p-hydroxybenzylamino)adenine produced only a slight decrease in root growth. BA-7-G and BA-9-G did not affect root growth.Abbreviations BA N ⁶-benzyladenine - iP isopentenyl-adenine - DHZ dihydrozeatin - BAR N ⁶-benzyladenosine - BAMP N ⁶-benzyladenosine 5-monophosphate - BA-7-G N ⁶-benzyladenine-7--d-glucopyranoside - BA-9-G N ⁶-benzyladenine-9--d-glucopyranoside - m-OH BA N ⁶-(m-hydroxybenzylamino)adenine - o-OH BA N ⁶-(o-hydroxybenzylamino)adenine - p-OH BA N ⁶-(p-hyrdoxybenzylamino)adenine - HPLC high performance liquid chromatography - gFW grams fresh weight     

Issues in using the WinRHIZO system to determine physical characteristics of plant fine roots☆

Image analysis systems have facilitated rapid measurements of fine root length (RL), diameter (RD), volume (RV), etc. The WinRHIZO system is unlike other image analysis systems in that it can detect, and make corrections for, areas of root overlap. It is designed to be capable of using both Regent’s non-statistical method (WinRHIZO method) and Tennant’s statistical method (line-intersect method), and can simultaneously output the root measurements by both methods when they are chosen at the same time. This study tested: (1), the efficacy of the overlap correction function in the WinRHIZO system; and (2), the consistency of fine root measurements between the WinRHIZO and Tennant methods with two sets of root measurement data from winter wheat (Triticum aestivium L.). The results showed that there were significant differences in RL, RD and RV between small root samples with and without stumps. The impact of root stumps outweighed the overlap correction efficacy in WinRHIZO. The values from the Tennant method are significantly different from those using the WinRHIZO method, although both results are statistically closely correlated. This indicated how critical it was to use without-stump root samples when using image analysis systems to measure RL, RD, RV, etc., and to keep in mind that a significant difference in root measurements may be methodologically related when comparing the results of various experiments from these two methods. Our research results bear important implications for the study of root ecology.     

Deuterium NMR of Raft Model Membranes Reveals Domain-Specific Order Profiles and Compositional Distribution

In this report, we applied site-specifically deuterated N-stearoylsphingomyelins (SSMs) to raft-exhibiting ternary mixtures containing SSM, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol (Chol) and successfully acquired deuterium quadrupole coupling profiles of SSM from liquid-ordered (L_o) and liquid-disordered (L_d) domains. To our knowledge, this is the first report that shows detailed lipid chain dynamics separately and simultaneously obtained from coexisting L_o and L_d domains. We also found that the quadrupole profile of the L_o phase in the ternary system was almost identical to that in the SSM-Chol binary mixture, suggesting that the order profile of the binary system is essentially applicable to more complicated membrane systems in terms of the acyl chain order. We also demonstrated that ²H NMR spectroscopy, in combination with organic synthesis of deuterated components, could be used to reveal the accurate mole fractions of each component distributed in the L_o and L_d domains. As compared with the reported tie-line analysis of phase diagrams, the merit of our ²H NMR analysis is that the domain-specific compositional fractions are directly attainable without experimental complexity and ambiguity. The accurate compositional distributions as well as lipid order profiles in ternary mixtures are relevant to understanding the molecular mechanism of lipid raft formation.     

Electrospray ionization mass spectroscopic analysis of peptides modified with N-ethylmaleimide or iodoacetanilide

The cysteine-specific modifiers we reported previously, N-ethylmaleimide (NEM) and iodoacetanilide (IAA), have been applied to label cysteine residues of peptides in combination with electrospray ionization mass spectrometry (ESI-MS/MS), and their scope in proteomic studies was examined. Peptides modified with N-ethylmaleimide (NEM) or iodoacetanilide (IAA) showed significant enhancement in ionization efficiencies. These modifiers were also found to remain intact in tandem mass spectrometry. Both combinations of N-ethylmaleimide (NEM) and d₅-N-ethylmaleimide (d₅-NEM), and iodoacetanilide (IAA) and ¹³C₆-iodoacetanilide (¹³C₆-IAA) were also shown to be applicable to quantitative analysis of a peptide.     

Dual Role of Prostaglandins (PGE2) in Regulation of Channel Density and Open Probability of Epithelial Na+ Channels in Frog Skin (R. pipiens)

Prostaglandins are important in signaling pathways involved in modulating the rates of Na⁺ transport in a diverse group of tissues possessing apical membrane epithelial channels. PGE₂ is known to cause either stimulation, inhibition or transient stimulatory changes of Na⁺ transport. We have continued our studies of frog skins that are known to respond to forskolin and PGE₂ with large steady-state increases of transport and have used noninvasive methods of blocker-induced noise analysis of Na⁺ channels to determine their channel densities (N _T ) and open probabilities (P _o ). In the absence of exogenous hormones, baseline rates of Na⁺ transport are especially high in scraped skins (R. pipiens pipiens) studied in the fall of the year. Na⁺ transport was inhibited by indomethacin and by removal of the unstirred layers of the corium (isolated epithelia) alone suggesting that PGE₂ is responsible for the sustained and elevated rates of transport in scraped skins. Changes of transport caused by indomethacin, forskolin or PGE₂ were unquestionably mediated by considerably larger changes of N _T than compensatory changes of P _o . Since cAMP caused no change of P _o in tissues pretreated with indomethacin, PGE₂ appears in this tissue to serve a dual role, increasing the steady state N _T by way of cAMP and decreasing P _o by unknown mechanisms. Despite appreciable PGE₂-related decreases of P _o , the net stimulation of transport occurs by a considerably greater cAMP-mediated increase of N _T . Received: 28 February 1996/Revised: 22 August 1996     

The synthesis and use of a 15N and 2H isotopically-labelled derivative of the spin-trap 3, 5-dibromo-4-nitrosobenzenesulphonic acid

Summary

We report the synthesis and use of d₂-¹⁵N isotopically-labelled 3, 5-dibromo-4-nitrosobenzenesulphonic acid (DBNBS-d₂-¹⁵N, as its sodium salt), a spin-trap possessing several advantages over non-labelled DBNBS. The simplification in the electron paramagnetic resonance spectra of radical adducts of DBNBS-d₂-¹⁵N compared with those of DBNBS not only results in increased sensitivity, but also facilitates the assignment and analysis of complex spectra. An example of this simplification is given.     

Effect of lactoferrin on oxidative features of ceruloplasmin

In our previous report we first described a complex between lactoferrin (Lf) and ceruloplasmin (Cp) with K _d ~ 1.8 μM. The presence of this complex in colostrum that never contains more than 0.3 μM Cp questions the reliability of K _d value. We carefully studied Lf binding to Cp and investigated the enzymatic activity of the latter in the presence of Lf, which allowed obtaining a new value for K _d of Cp–Lf complex. Lf interacting with Cp changes its oxidizing activity with various substrates, such as Fe²⁺, o-dianisidine (o-DA), p-phenylenediamine (p-PD) and dihydroxyphenylalanine (DOPA). The presence of at least two binding sites for Lf in Cp molecule is deduced from comparison of substrates’ oxidation kinetics with and without Lf. When Lf binds to the first site affinity of Cp to Fe²⁺ and to o-DA increases, but it decreases towards DOPA and remains unchanged towards p-PD. Oxidation rate of Fe²⁺ grows, while that of o-DA, p-PD and DOPA goes down. Subsequent Lf binding to the second center has no effect on iron oxidation, hampers DOPA and o-DA oxidation, and reduces affinity towards p-PD. Scatchard plot for Lf sorbing to Cp-Sepharose allowed estimating K _d for Lf binding to high-affinity (~13.4 nM) and low-affinity (~211 nM) sites. The observed effect of Lf on ferroxidase activity of Cp is likely to have physiological implications.     

Evidence of pores and thinned lipid bilayers induced in oriented lipid membranes interacting with the antimicrobial peptides, magainin-2 and aurein-3.3

Dynamic structures of supramolecular lipid assemblies, such as toroidal pores and thinned bilayers induced in oriented lipid membranes, which are interacting with membrane-acting antimicrobial peptides (AMPs), magainin-2 and aurein-3.3, were explored by ³¹P and ²H solid-state NMR (ssNMR) spectroscopy. Various types of phospholipid systems, such as POPC-d₃₁, POPC-d₃₁/POPG, and POPC-d₃₁/cholesterol, were investigated to understand the membrane disruption mechanisms of magainin-2 and aurein-3.3 peptides at various peptide-to-lipid (P:L) ratios. The experimental lineshapes of anisotropic ³¹P and ²H ssNMR spectra measured on these peptide-lipid systems were simulated reasonably well by assuming the presence of supramolecular lipid assemblies, such as toroidal pores and thinned bilayers, in membranes. Furthermore, the observed decrease in the anisotropic frequency span of either ³¹P or ²H ssNMR spectra of oriented lipid bilayers, particularly when anionic POPG lipids are interacting with AMPs at high P:L ratios, can directly be explained by a thinned membrane surface model with fast lateral diffusive motions of lipids. The spectral analysis protocol we developed enables extraction of the lateral diffusion coefficients of lipids distributed on the curved surfaces of pores and thinned bilayers on a few nanometers scale.     

Quantitative traits associated with adaptation of three barley (Hordeum vulgare L) cultivars to suboptimal iron supply

A laboratory method was developed that allows determination of in situ net nitrification with high sensitivity and at high temporal resolution. Nitrate in soils is quantitatively converted into nitrous oxide under strictly anaerobic conditions in the presence of 10 kPa acetylene by the soil endogenous denitrifier population, with the N₂O detected by a gas chromatograph equipped with a ⁶³Ni electron capture detector. Thus, even low net nitrification rates, i.e. small net increases in soil nitrate concentrations can easily be detected. Comparison of results using this method with results obtained using the classical in situ incubation method (buried bag soil incubation) revealed excellent agreement. Application of the new method allowed both determination of the seasonal pattern of net nitrification as well as correlation analysis between in situ NO and N₂O flux rates and in situ net nitrification rates of the forest soils studied. Regardless of the forest site studied (spruce, spruce limed, beech), and during each year of a 3 years period (1995–1997), net nitrification varied strongly with season and was least during winter and greatest during summer. The long-term annual, mean rate of net nitrification for the untreated spruce site, the limed spruce site and the beech site were 1.54 ± 0.27 mg N kg^–1 sdw d^–1, 1.92 ± 0.23 mg N kg^–1 sdw d^–1 and 1.31 ± 0.23 mg N kg^–1 sdw d^–1, respectively. In situ rates of nitrification and NO and N₂O emission were strongly correlated for all sites suggesting that nitrification was the dominate source of NO as well as N₂O.     

Structural determination of the O-antigenic polysaccharide from Escherichia coli O74

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O74 has been determined. Component analysis, together with ¹H and ¹³C NMR spectroscopy as well as ¹H,¹⁵N-HSQC experiments were employed to elucidate the structure. Inter-residue correlations were determined by ¹H,¹H-NOESY and ¹H,¹³C-heteronuclear multiple-bond correlation experiments. The PS is composed of tetrasaccharide repeating units with the following structure:

Full-size image (5K)

Cross-peaks of low intensity from an α-linked N-acetylglucosamine residue were present in the NMR spectra, and spectral analysis indicates that they originate from the penultimate residue in the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-glucosamine residue at its reducing end. The ¹H, ¹³C and ¹⁵N NMR chemical shifts of the α- and β-anomeric forms of d-Fucp3NAc are also reported. The repeating unit of the E. coli O74 O-antigen is identical to that of the capsular polysaccharide from E. coli K45.     

Automated image analyses for separating plant roots from soil debris elutrated from soil cores

Historically, destructive root sampling has been labor intensive and requires manual separation of extraneous organic debris recovered along with the hydropneumatic elutriation method of separating plant roots from soils. Quantification of root system demographics by public domain National Institute of Health (NIH-Image) and Root Image Processing Laboratory (RIPL) image processing algorithms has eliminated much of the labor-intensive manual separation. This was accomplished by determining the best length to diameter ratio for each object during image analyses. Objects with a length to diameter ratio less than a given threshold are considered non-root materials and are rejected automatically by computer algorithms. Iterative analyses of length to diameter ratios showed that a 15:1 ratio was best for separating images of maize (Zea mays L.) roots from associated organic debris. Using this threshold ratio for a set of 24 soil cores, a highly significant correlation (r² = 0.89) was obtained between computer image processed total root length per core and actual root length. A linear relationship (r² = 0.80) was observed between root lengths determined by NIH-Image analyses and lengths determined independently by the RIPL imaging system, using the same maize root + debris samples. This correlation demonstrates that computer image processing provides opportunities for comparing root length parameters between different laboratories for samples containing debris.