The application of atomic force microscopy to topographical studies and force measurements on the secreted adhesive of the green alga Enteromorpha

Atomic force microscopy (AFM) enables the topographical structure of cells and biological materials to be resolved under natural (physiological) conditions, without fixation and dehydration artefacts associated with imaging methods in vacuo. It also provides a means of measuring interaction forces and the mechanical properties of biomaterials. In the present study, AFM has been applied for the first time to the study of the mechanical properties of a natural adhesive produced by a green plant cell. Swimming spores of the green alga Enteromorpha linza (L.) J. Ag. (7–10 μm) secrete an adhesive glycoprotein which provides firm anchorage to the substratum. Imaging of the adhesive in its hydrated state revealed a swollen gel-like pad, approximately 1 μm thick, surrounding the spore body. Force measurements revealed that freshly released adhesive has an adhesion strength of 173 ± 1.7 mN m⁻¹ (mean ± SE; n=90) with a maximum value for a single adhesion force curve of 458 mN m⁻¹. The adhesive had a compressibility (equivalent to Young's modulus) of 0.54 × 10⁶ ± 0.05 × 10⁶ N m⁻² (mean ± SE; n=30). Within minutes of release the adhesive underwent a progressive `curing' process with a 65% reduction in mean adhesive strength within an hour of settlement, which was also reflected in a reduction in the average length of the adhesive polymer strands (polymer extension) and a 10-fold increase in Young's modulus. Measurements on the spore surface itself revealed considerably lower adhesion-strength values but higher polymer-extension values than the adhesive pad, which may reflect the deposition of different polymers on this surface as a new cell wall is formed. The study demonstrates the value of AFM to the imaging of plant cells in the absence of fixation and dehydration artefacts and to the characterisation of the mechanical properties of plant glycoproteins that have potential utility as adhesives. Received: 22 February 2000 / Accepted: 20 April 2000     

BENDING STIFFNESS OF CYLINDRICAL PLANT ORGANS WITH A ‘CORE-RIND’ CONSTRUCTION: EVIDENCE FROM JUNCUS EFFUSUS LEAVES

The mechanical behavior of leaves of Juncus effusus L. in bending was investigated in terms of a closed-form analytical solution derived to predict the bending stiffness of a cylindrical sandwich beam consisting of an outer ‘rind’ (sclerenchyma and chlorenchyma) and an inner ‘core’ (aerenchyma). The elastic moduli (E_TOTAL) of intact leaves was measured by means of multiple resonance frequency spectra and compared to that of leaves for which the aerenchymatous core was surgically destroyed. Based on ten leaves, E_TOTAL = 22.33 × 10⁴ ± 5.37 ± 10⁴ kg · cm^–2 while the elastic modulus of the ‘rind’ was 22.29 × 10⁴ ± 5.69 × 10⁴ kg · cm^–2. The elastic modulus of the ‘core’ was estimated at 3.12 × 10⁴ ± 1.42 × 10⁴ kg · cm^–2. Load-deflection curves for three leaf segments indicated leaves were linearly elastic within the range of loading and could be predicted with considerable accuracy based on the closed-form solution. The aerenchymatous core was found to contribute very little to the bending stiffness of leaves, although its contribution appeared to increase as leaf diameter decreased. Leaves mechanically failed by Brazier buckling when excessively loaded and were best considered to mechanically operate as hollow tubes. Nonetheless, the analytical solution for bending stiffness could be applied and, in theory, can be used to predict the behavior of other plant organs with a ‘corerind’ construction.     

Characterization and Regulatory Properties of Glucose-6-Phosphate Dehydrogenase from Black Gram (Phaseolus mungo)

Glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) was partially purified by fractionation with ammonium sulfate and phosphocellulose chromatography. The K_m value for glucose-6-phosphate is 1.6 × 10^?4 and 6.3 × 10^?4M at low (1.0–6.0 × 10^?4M) and high (6.0–30.0 × 10^?4M) concentrations of the substrate, respectively. The K_m value for NADP⁺ is 1.4 × 10^?5M. The enzyme is inhibited by NADPH, 5-phosphoribosyl-1-pyrophosphate, and ATP, and it is activated by Mg²⁺, and Mn²⁺. In the presence of NADPH, the plot of activity vs. NADP⁺ concentration gave a sigmoidal curve. Inhibition of 5-phosphoribosyl-1-pyrophosphate and ATP is reversed by Mg²⁺ or a high pH. It is suggested that black gram glucose-6-phosphate dehydrogenase is a regulatory enzyme of the pentose phosphate pathway.     

Dynamic stiffness and crossbridge action in muscle

Small sinusoidal vibrations at 300 Hz were applied to frog sartorius muscle to measure the dynamic stiffness (Young's modulus) throughout the course of tetanus. For a peak-to-peak amplitude of 0.4% the dynamic Young's modulus increased from 1.5×10⁵ Nm^–2 in the resting state to 2×10⁷ Nm^–2 in tetanus. After correction for the external connective tissue, the dynamic Young's modulus of the muscle was almost directly proportional to the tension throughout the development of tetanus. The ratio of dynamic Young's modulus to tensile stress thus remained constant (with a value at 300 Hz of approximately 100), consistently with Huxley and Simmons' identification of the crossbridges as the source of both tension and stiffness.For a single crossbridge the ratio of stiffness to tension was 8.2×10⁷ m^–1 at 300 Hz; it is deduced from literature data that the limiting value at high frequencies is about 1.6×10⁸ m^–1. This ratio is interpreted on Harrington's (1971) model to show that crossbridge action can be explained by a helix-coil transition of about 80 out of the 260 residues in each S-2 myosin strand. It is also shown that a helix-coil model can account for the observed rapid relaxation of muscle without invoking any complex behaviour of the crossbridge head.     

Mechanism of Catechin Chemiluminescence in the Presence of Active Oxygen

The photon emission (chemiluminescence; CL) of catechin in the presence of active oxygen species (hydrogen peroxide, hydroxyl radical tert-butyl hydroperoxide and tert-butyl oxyl radical) and acetaldehyde was confirmed to occur non-enzymatically at room temperature in aqueous neutral conditions. The CL intensity [P] in the presence of active oxygen species (X), catalytic species (Y) and receptors (Z) is predicted by [P] = k [X] [Y] [Z]. The calculated photon constants (k) of 8 catechins and gallic acid were 8.23 × 10⁶ M⁻² s⁻¹ counts ((−)-epigallocatechin), 2.78 × 10⁶ ((−)-epigallocatechin gallate), 4.66 × 10⁵ ((−)-gallocatechin gallate), 4.36 × 10⁵ ((−)-gallocatechin), 2.70 × 10⁵ ((−)-epicatechin), 6.44 × 10⁴ ((−)-catechin), 5.85 × 10⁴ ((−)-epicatechin gallate), 4.78 × 10⁴ (gallic acid) and 3.54 × 10⁴ ((−)-catechin gallate), respectively. The system of active oxygen species, catalytic species and receptors is proposed to be a scavenging mechanism for active oxygen species. In the presence of acetaldehyde, (−)-epigallocatechin (maximum k value among catechins tested) reacted with tert-BuOOH to form tert-BuOH as determined by HPLC analysis.     

Cell density dependent response of E. Coli cells to weak ELF magnetic fields

The effects of weak magnetic fields of extremely low frequency (ELF) on E. coli K12 AB1157 cells were studied by the method of anomalous viscosity time dependencies (AVTD). E. coli cells at different densities within a range of 5 × 10⁵–10⁹ cell/ml were exposed to ELF (sinusoidal, 30 μT peak, 15 min) at a frequency of 9 Hz. A transient effect with maximum 40–120 min after exposure was observed. Kinetics of the per-cell-normalised ELF effects fitted well to a Gaussian distribution for all densities during exposure. A maximum value of these kinetics and a time for this maximum were strongly dependent on the cell density during exposure. These data suggest a cell-to-cell interaction during response to ELF. Both dependencies had three regions close to a plateau within the ranges of 3 × 10⁵ − 2 × 10⁷ cell/ml, 4 × 10⁷ − 2 × 10⁸ cell/ml and 4 × 10⁸–10⁹ cell/ml and two rather sharp transitions between these plateaus. The effect reached a maximum value at a density of 4 × 10⁸ cell/ml. Practically no effect was observed at the lowest density of 3 × 10⁵ cell/ml. The data suggested that the ELF effect was mainly caused by a secondary rather than a primary reaction. The filtrates from exposed cells neither induced significant AVTD changes in unexposed cells nor increased the ELF effect when were added to cells before exposure. The data did not provide evidence for significant contribution of stable chemical messengers, but some unstable compounds such as radicals could be involved in the mechanism of cell-to-cell interaction during response to ELF. The results obtained were also in accordance with a model based on an re-emission of secondary photons during resonance fluorescence. Bioelectromagnetics 19:300–309, 1998. © 1998 Wiley-Liss, Inc.     

PROPERTIES OF TYROSINE HYDROXYLASE IN PERIPHERAL NERVES

Approximately 80 per cent of tyrosine hydroxylase activity in bovine mandibular nerve and rabbit sciatic nerve was soluble, and the rest of the activity was particle-bound. The soluble enzyme in bovine mandibular nerve was isolated by ammonium sulphate fractionation (25–35 per cent saturation). The enzyme had a pH optimum at 5·9 in Tris-acetate buffer, and at 6·5 in Tris-HCl or phosphate buffer. The enzyme required a tetrahydropteridine cofactor. K_m values toward various tetrahydropteridines such as l -erythro-tetrahydrobiopterin (a probable natural cofactor), 2-amino-4-hydroxy-6-methyltetrahydropteridine, and 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine were 2 × 10⁻⁵m , 5 × 10⁻⁵m and 4 × 10⁻⁴m , respectively. The K_m value for tyrosine at 1 × 10⁻³m -2-amino-4-hydroxy-6-methyltetrahydropteridine as a cofactor was 5 × 10⁻⁵m . The enzyme activity was markedly stimulated with Fe²⁺ or catalase, but Fe²⁺ gave higher activity. The activity was inhibited with α, α′-dipyridyl, l -α-methyl-p-tyrosine, and various catecholamines. Among catecholamines, dopamine was the most potent inhibitor. l -5-Hydroxytryptophan was an inhibitor as potent as dopamine. Neither d -5-hydroxytryptophan nor 5-hydroxytryptamine inhibited the enzyme. The inhibition by l -5-hydroxytryptophan was partially competitive with tetrahydrobiopterin at concentrations higher than 9 × 10⁻⁵m , and partially uncompetitive at concentrations lower than 9 × 10⁻⁵m . The addition of heparin or lysolecithin did not affect enzyme activity with tetrahydrobiopterin as cofactor.     

Profile of common prostate cancer risk variants in an unscreened Romanian population

To find sequence variants affecting prostate cancer (PCA) susceptibility in an unscreened Romanian population we use a genome‐wide association study (GWAS). The study population included 990 unrelated pathologically confirmed PCA cases and 1034 male controls. DNA was genotyped using Illumina SNP arrays, and 24.295.558 variants were imputed using the 1000 Genomes data set. An association test was performed between the imputed markers and PCA. A systematic literature review for variants associated with PCA risk identified 115 unique variants that were tested in the Romanian sample set. Thirty of the previously reported SNPs replicated (P‐value < 0.05), with the strongest associations observed at: 8q24.21, 11q13.3, 6q25.3, 5p15.33, 22q13.2, 17q12 and 3q13.2. The replicated variants showing the most significant association in Romania are rs1016343 at 8q24.21 (P = 2.2 × 10^?4), rs7929962 at 11q13.3 (P = 2.7 × 10^?4) and rs9364554 at 6q25.2 (P = 4.7 × 10^?4). None of the variants tested in the Romanian GWAS reached genome‐wide significance (P‐value <5 × 10^?8) but 807 markers had P‐values <1 × 10^?4. Here, we report the results of the first GWAS of PCA performed in a Romanian population. Our study provides evidence that a substantial fraction of previously validated PCA variants associate with risk in this unscreened Romanian population.     

Studies on the myosin molecule from smooth muscle

The myosin molecule was extracted from the smooth muscle parts of horse esophagus and purified by ammonium sulfate fractionation. The schlieren pattern of the sedimentation velocity run showed a very sharp single peak of.5.9. S (s_20,w). Molecular weight of the protein was measured by means of the Archibald and sedimentation equilibrium methods, both in 0.5M KCI buffered by 1/150 M phosphate at pH 7.5 and at 5°C. The values obtained were 6.25 × 10⁵ and 5.81 × 10⁵respectively, for the two methods. The second virial coefficients were 1.1 × 10⁴ and 1.2 × 10^?4 ml/g. Denatured smooth muscle myosin was prepared in a solution of 5M guanidine HC1 containing 0.4 M KC1 and 0.2 M β-mercaptoet hanol buffered at p^H 8.0. The weight-average molecular weight of the denatured smooth muscle myosin was 2.24 × 10⁵ and the second virial coefficient was 7.6 × 10^?4 ml/g. The values described above are in good agreement with those reported for rabbit skeletal myosin with ammonium sulfate fractionation. The molecular dimension of the molecule is estimated as the value for an axial ratio of 100, assuming a rigid rod molecular model for this molecule, both the thermodynamical and hydrodynamical treatment being in a good agreement with this estimation.     

Determination of corneal gel dynamics

From observations of the dynamics of light scattered by the cornea, intensity autocorrelation func-tions that revealed two independent diffusion coefficients, D (fast) = 2.4±0.2×10^–7 cm²/s and D (slow) = 9.4±1.3× 10^–9 cm²/s, were obtained. The diffusion coefficients were found to be statistically independent of the position and depth on the lateral surface of the cornea from which the scattered light was sampled. The slow diffusion coefficients obtained from light sampled from within cross-sections of the cornea were, however, measurably different. Diffusion coefficients obtained independently from observations of the kinetics of corneal swelling for comparison were found to be several orders of magnitude greater than those obtained from light scattering. The large disparity in the diffusion coefficients obtained from the two independent methods invoked the possibility that the lamellar layers within the cornea behave as individual gel sheets. Irrespective of this additional hypothesis, divergent behavior in the measured total scattered light intensities and diffusion coefficients upon varying external conditions, such as temperature or pressure (stretching), was observed. Namely, a slowing down of the dynamic modes accompanied by increased “static” scattered light intensities was observed. Although the slowing down of the dynamic modes is possibly indicative of the reduced affinity of protein binding to the gel matrix that “softens” the gel, the divergent behavior in the scattered light intensities and diffusion coefficients is, however, more characteristic of a phase transition. In addition, the divergent behavior in the scattered light intensities and diffusion coefficients was reversible up to a critical temperature (∼55 °C) or stretching (∼16%). Received: 18 March 1998 / Revised version: 4 February 1999 / Accepted: 4 February 1999     

Calorific values of Chlorella vulgaris (Trebouxiophyceae) as a function of different phosphorus concentrations

Quantification of the calorific content of microalgae is critical in studies of energy flow, trophic partitioning, plant/herbivore interactions in aquaculture and biomass production for biofuels. We investigated the calorific value and biochemical composition of Chlorella vulgaris at different phosphorus (P) concentrations (6.0 × 10^?7, 2.3 × 10^?6 and 2.3 × 10^?4 mol L^?1 P). As expected, the control (2.3 × 10^?4 mol L^?1 P) supported better growth than P limited treatments. Biomolecules like total carbohydrates and lipids accumulated under P limitation, which significantly correlated with high calorific values. Lipid class composition showed that triacylglycerols were the most accumulated under P limited conditions. The calorific value reported under control conditions (13.78 kJ g^?1) was less than those obtained under P limitation (30.47–33.07 kJ g^?1). The highest calorific value with less growth retardation was obtained at 2.3 × 10^?6 mol L^?1 P.     

Effects of nitrogen nutrition and root medium water potential on growth, nitrogen uptake and osmotic adjustment of rice

The effects of nitrogen (N) nutrition on growth, N uptake and leaf osmotic potential of rice plants (Oryza sativa L. ev. IR 36) during simulated water stress were determined. Twenty-one-day-old seedlings in high (28.6 × 10 ^?4M) and low (7.14 × 10 ⁴M) N levels were exposed to decreased nutrient solution water potentials by addition of polyethylene glycol 6000. The roots were separated from the solution by a semi-permeable membrane. Nutrient solution water potential was ?0.6 × 10⁵ Pa and was lowered stepwise to ?1 × 10⁵, ?2 × 10⁵, ?4 × 10⁵ and ?6 × 10⁵ Pa at 2-day intervals. Plant height, leaf area and shoot dry weight of high and low nitrogen plants were reduced by lower osmotic potentials of the root medium. Osmotic stress caused greater shoot growth reduction in high N than in low N plants. Stressed and unstressed plants in 7.14 × 10⁴M N had more root dry matter than the corresponding plants in 28.6 × 10⁴M N. Dawn leaf water potential of stressed plants was 1 × 10⁵ to 5.5 × 10⁵ Pa lower than nutrient solution water potential. Nitrogen-deficient water-stressed plants, however, maintained higher dawn leaf water potential than high nitrogen water-stressed plants. It is suggested that this was due to higher root-to-shoot ratios of N deficient plants. The osmotic potentials of leaves at full turgor for control plants were about 1.3 × 10⁵ Pa higher in 7.14 × 10^?4M than in 28.6 × 10^?4M N and osmotic adjustment of 2.6 × 10⁵ and 4.3 × 10⁵ Pa was obtained in low and high N plants, respectively. The nitrogen status of plants, therefore, affected the ability of the rice plant to adjust osmotically during water stress. Plant water stress decreased transpiration and total N content in shoots of both N treatments. Reduced shoot growth as a result of water stress caused the decrease in amount of water transpired. Transpiration and N uptake were significantly correlated. Our results show that nitrogen content is reduced in water-stressed plants by the integrated effects of plant water stress per se on accumulation of dry matter and transpiring leaf area as well as the often cited changes in soil physical properties of a drying root medium.     

The Characterization of Rennin Action on κ-Casein Using CM-Cellulose

Rennin action on κ-Casein was studied using the CM-cellulose method which determines the amount of para-κ-casein formed during the enzymatic hydrolysis of κ-casein. The reaction rate was measured as a function of the time and enzyme concentration. A Km value of 8.9 ×10^?4m and a V value of 1.2 × 10^?4 M/min were obtained under the assay condition used in this study. The maximum initial rate of para-κ-casein formation occurred at pH 5.0 and 50°C. The present study also demonstrated that the CM-cellulose method is useful for measuring the rennin activity on κ-casein.     

Peroxyoxalate chemiluminescent assay for oxidase activities based on detecting enzymatically formed hydrogen peroxide

A sensitive peroxyoxalate chemiluminescent (PO-CL) assay for activities of oxidases (uricase, choline oxidase, cholesterol oxidase and xanthine oxidase) which catalyse a formation of hydrogen peroxide was developed using 4,4′-oxalyl-bis[(trifluoromethylsulphonyl)imino]trimethylene-bis(4-methylmorpholinium)trifluoromethanesulphonate as a chemiluminogenic reagent and 2,4,6,8-tetramorpholinopyrimido[5,4-d]pyrimidine as a fluorophore. The standard curve for hydrogen peroxide was linear over the range 1 × 10^?7-1 × 10^?4 mol/L. Relative standard deviations for oxidase assays were 5.1–12.7% (n = 10). Detection limits were 1 × 10^?3 U/mL for uricase, 5 × 10^?4 U/mL for choline oxidase, 5 × 10^?3 U/mL for cholesterol oxidase and 5 × 10^?4 U/mL xanthine oxidase (sample to blank ratio, 3).     

Kinetics of Water Transport in Eel Intestinal Vesicles

Brush border membrane vesicles, BBMV, from eel intestinal cells or kidney proximal tubule cells were prepared in a low osmolarity cellobiose buffer. The osmotic water permeability coefficient P _f for eel vesicles was not affected by pCMBS and was measured at 1.6 × 10⁻³ cm sec⁻¹ at 23°C, a value lower than 3.6 × 10⁻³ cm sec⁻¹ exhibited by the kidney vesicles and similar to published values for lipid bilayers. An activation energy E _a of 14.7 Kcal mol⁻¹ for water transport was obtained for eel intestine, contrasting with 4.8 Kcal mol⁻¹ determined for rabbit kidney proximal tubule vesicles using the same method of analysis. The high value of E _a , as well as the low P _f for the eel intestine is compatible with the absence of water channels in these membrane vesicles and is consistent with the view that water permeates by dissolution and diffusion in the membrane. Further, the initial transient observed in the osmotic response of kidney vesicles, which is presumed to reflect the inhibition of water channels by membrane stress, could not be observed in the eel intestinal vesicles. The P _f dependence on the tonicity of the osmotic shock, described for kidney vesicles and related to the dissipation of pressure and stress at low tonicity shocks, was not seen with eel vesicles. These results indicate that the membranes from two volume transporter epithelia have different mechanisms of water permeation. Presumably the functional water channels observed in kidney vesicles are not present in eel intestine vesicles. The elastic modulus of the membrane was estimated by analysis of swelling kinetics of eel vesicles following hypotonic shock. The value obtained, 0.79 × 10⁻³ N cm⁻¹, compares favorably with the corresponding value, 0.87 × 10⁻³ N cm⁻¹, estimated from measurements at osmotic equilibrium. Received: 28 January 1999/Revised: 15 June 1999     

Differential in vitro inhibition of polyphenoloxidase from a wild edible mushroom Lactarius salmonicolor

The polyphenol oxidase (LsPPO) from a wild edible mushroom Lactarius salmonicolor was purified using a Sepharose 4B-L-tyrosine-p-amino benzoic acid affinity column. At the optimum pH and temperature, the K_M and V_Max values of LsPPO towards catechol, 4-methylcatechol and pyrogallol were determined as 0.025 M & 0.748 EU/mL, 1.809 × 10^{? 3} M & 0.723 EU/mL and 9.465 × 10^{? 3} M & 0.722 EU/mL, respectively.

Optimum pH and temperature values of LsPPO for the three substrates above ranged between the pH 4.5–11.0 and 5–50°C. Enzyme activity decreased due to heat denaturation with increasing temperature. Effects of a variety of classical PPO inhibitors were investigated opon the activity of LsPPO using catechol as the substrate. IC₅₀ values for glutathione, p-aminobenzenesulfonamide, L-cysteine, L-tyrosine, oxalic acid, β-mercaptoethanol and syringic acid were determined as 9.1 × 10^{? 4}, 2.3 × 10^{? 4} M, 1.5 × 10^{? 4} M, 3.8 × 10^{? 7} M, 1.2 × 10^{? 4} M, 4.9 × 10^{? 4} M, and 4 × 10^{? 4} M respectively. Thus L-tyrosine was by far the most effective inhibitor. Interestingly, sulfosalicylic acid behaved as an activator of LsPPO in this study.     

Properties of glutamate dehydrogenase from Beta vulgaris

Glutamate-NAD oxidoreductase, E.C. 1.4.1.3 (GDH), from seedlings of Beta vulgaris cv. Rota, Jahnsch Peragis Comp., was enzymatically characterized. This enzyme with molecular weight of 2.6 × 10⁵ has a pH optimum of around 8 for animation of α-KGA and around 9.5 for the desamination of glutamate. The apparent K_m for α-KGA is 6.7 × 10^?4M, for NH₃ 2.5 × 10^?3M, for NADH 3.2 × 10^?5M and for NAADPH 5.5 × 10^?4M. NAD¹ inhibits the reaction non-competitively when NADPH serves as substrate. The apparent K¹ is 4.5 × 10^?4M. The data are discussed on relation to the properties of GDH from other plant sources.     

A novel LncRNA-based prognostic score reveals TP53-dependent subtype of lung adenocarcinoma with poor survival

The prognostic signatures play an essential role in the era of personalised therapy for cancer patients including lung adenocarcinoma (LUAD). Long noncoding RNA (LncRNA), a relatively novel class of RNA, has shown to play a crucial role in all the areas of cancer biology. Here, we developed and validated a robust LncRNA-based prognostic signature for LUAD patients using three different cohorts. In the discovery cohort, four LncRNAs were identified with 10% false discovery rate and a hazard ratio of >10 using univariate Cox regression analysis. A risk score, generated from the four LncRNAs’ expression, was found to be a significant predictor of survival in the discovery and validation cohort (p = 9.97 × 10 ⁻⁸ and 1.41 × 10 ⁻³, respectively). Further optimisation of four LncRNAs signature in the validation cohort, generated a three LncRNAs prognostic score (LPS), which was found to be an independent predictor of survival in both the cohorts ( p = 1.00 × 10 ⁻⁶ and 7.27 × 10 ⁻⁴, respectively). The LPS also significantly divided survival in clinically important subsets, including Stage I ( p = 9.00 × 10 ⁻⁴ and 4.40 × 10 ⁻², respectively), KRAS wild-type (WT), KRAS mutant ( p = 4.00 × 10 ⁻³ and 4.30 × 10 ⁻², respectively) and EGFR WT ( p = 2.00 × 10 ⁻⁴). In multivariate analysis LPS outperformed, eight previous prognosticators. Further, individual members of LPS showed a significant correlation with survival in microarray data sets. Mutation analysis showed that high-LPS patients have a higher mutation rate and inactivation of the TP53 pathway. In summary, we identified and validated a novel LncRNA signature LPS for LUAD.     

Hydrodynamic properties of mucins secreted by primary cultures of Guinea-pig tracheal epithelial cells: determination of diffusion coefficients by analytical ultracentrifugation and kinetic analysis of mucus gel hydration and dissolution

We have used two different approaches to determine hydrodynamic parameters for mucins secreted by guinea-pig tracheal epithelial cells in primary culture. Cells were cultured under conditions that promote mucous cell differentiation. Secreted mucins were isolated as the excluded fraction from a Sepharose CL-4B gel filtration column run under strongly dissociating conditions. Biochemical analysis confirmed the identity of the high molecular weight material as mucins. Analytical ultracentrifugation was used to study the physical properties of the purified mucins. The weight average molecular mass (M _w ) for three different preparations ranged from 3.3×10⁶ to 4.7×10⁶ g/mol (corresponding to an average structure of 1 – 2 subunits), and the sedimentation coefficient from 25.5 to 35 S. Diffusion coefficients ranging from 4.5×10^–8 to 6.4×10^–8 cm²/s were calculated using the Svedberg equation. A polydispersity index (M _z /M _w ) of ∼1.4 was obtained. Diffusivity values were also determined by image analysis of mucin granule exocytosis captured by videomicroscopy. The time course of hydration and dissolution of mucin was measured and a relationship is presented which models both phases, each with first order kinetics, in terms of a maximum radius and rate constants for hydration and dissolution. A median diffusivity value of 8.05×10^–8 cm²/s (inter-quartile range = 1.11×10^–7 to 6.08×10^–8 cm²/sec) was determined for the hydration phase. For the dissolution phase, a median diffusivity value of 6.98×10^–9 cm²/s (inter-quartile range = 1.47×10^–8 to 3.25×10^–9 cm²/sec) was determined. These values were compared with the macromolecular diffusion coefficients (D _20,w ) obtained by analytical ultracentrifugation. When differences in temperature and viscosity were taken into account, the resulting D _37,g was within the range of diffusivity values for dissolution. Our findings show that the physicochemical properties of mucins secreted by cultured guinea-pig tracheal epithelial cells are similar to those of mucins of the single or double subunit type purified from respiratory mucus or sputum. These data also suggest that measurement of the diffusivity of dissolution may be a useful means to estimate the diffusion coefficient of mucins in mucus gel at the time of exocytosis from a secretory cell. Received: 10 March 1998 / Accepted: 27 March 1998     

Simultaneous Measurement of NH(4) Absorption and N(2) Fixation by Glycine max L. : RESPONSE TO TEMPERATURE, pH, AND EXTERNAL NITROGEN CONCENTRATION

Curvature, bending moment, and second moment of stem cross-sectional area were evaluated from photographic data and used to compute flexural rigidity and Young's modulus in the panicle rachis of rice, Oryza sativa L. `M-101.' Flexural rigidity C, and its components E, Young's modulus, and I, the moment of inertia of the area about the neutral axis, were evaluated 1.5 cm (tip), 9.5 cm (mid), and 16.5 cm (base) from the tip of the panicle rachis. In dynes per square centimeter, C increases from 1.1 × 10³ near the tip to 1.09 × 10⁴ in the middle to 5.35 × 10⁴ in the basal region of the rachis. Of the components of C, the I changes have the larger effect, increasing from 2.12 × 10⁻⁷ centimeters⁴ near the tip to 8.21 × 10⁻⁷ centimeters⁴ in mid regions to 6.0 × 10⁻⁶ centimeters⁴ in the basal regions. Young's modulus increases from 4.8 × 10⁹ dynes per square centimeter near the tip to 1.4 × 10¹⁰ dynes per square centimeter in mid regions then falls to 7.4 × 10⁹ dynes per square centimeter near the base of the main stem. Values of Young's modulus from Instron experiments were in satisfactory agreement with values calculated from the beam bending equation. Flexural rigidity in the curved region of the panicle proved independent of panicle load, indicating that the dissected panicle rachis behaves in some respects as a tapered loaded beam.