Analysis of diffusion and relaxation behavior of water in apple parenchymal cells

It has been demonstrated by an example of apple parenchymal cells that NMR spectroscopy can be used to analyze the relaxation and diffusion of water molecules in plant cells. With small diffusion times, three relaxation components have been distinguished, which correspond to water in a vacuole, in the cytoplasm, and in intercellular liquid. The coefficient of self-diffusion corresponding to these components have been determined. With large diffusion times, it is possible to distinguish two components. For the slowly relaxing component (which corresponds to water in a vacuole), the regime of restricted diffusion was observed. For a quickly relaxing component, an anomalous increase in the coefficient of self-diffusion with the time of diffusion took place.     

Relationship between leaf water spin-lattice relaxation time and water relation parameters in three wheat cultivars

Pot culture studies were conducted using two drought-tolerant and one susceptible cultivar of wheat (Triticum aestivum L.) under three different moisture regimes. Proton spin-lattice relaxation time, T₁, leaf water content, LWC, leaf water potential, Ψ, solute potential, Ψ_s and turgor potential, Ψ_p were measured from 45 to 75 d after sowing at weekly intervals. The three cultivars did not differ significantly in their values of LWC, leaf water potential, and their components in the stressed and unstressed plants; but they did differ significantly in their T₁ values both under stressed and unstressed conditions on all days of measurement, with the drought tolerant cultivars having a higher T₁ compared to the susceptible cultivar. This suggests that leaf water T₁ is a better parameter for describing plant water status than the traditional water relation indices. The relation between Ψ and T₁ was logarithmic, indicating the similarity between T₁ and water activity of the cellular water.     

NMR relaxation study of water protons in Syrian hamster fetal cells at 300 MHz

TheT ₁ andT ₂ relaxation times of water protons in two cell types in culture derived from Syrian hamster fetuses (normal primary or secondary fetal cells vs BP6T tumor cells derived from the normal cells transformed by carcinogens) were measured at 7.05 Tesla magnetic field (proton frequency =300 MHz). TheT ₁/T ₂ ratios and the correlation time, τ _c , calculated from theT ₁/T ₂ ratio of cellular water protons, are significantly different in these two fibroblastic cell types of the same biological origin and with similar morphologies and growth rates in culture.     

State of water in starch-water systems in the gelatinization temperature range as investigated using dielectric relaxation spectroscopy

The dielectric response of native wheat starch-water slurries containing 5-60% starch (w/w) was measured in the frequency range of 0.2-20 GHz after heating the slurries to 7 different temperatures between 25 and 90 °C for 30 min. Three relaxations, with relaxation time range of 4-9 ps, 20-25 ps and 230-620 ps at 25 °C, were identified from the dielectric spectra of starch slurries. The fastest relaxation process (4-9 ps) was attributed to bulk water while the two slower relaxations were attributed to the confined water molecules present in the starch-water system. The amount of water exhibiting the slowest relaxation (230-620 ps) was calculated to be 0.08-0.16 g water/g starch, which was close to the monolayer water associated with wheat starch. Mobility of bulk water was significantly reduced (P < 0.001) upon gelatinization at low starch concentration (10% starch), but remained unaffected at higher starch concentrations. The mobility of two slower relaxing water species was not significantly influenced (P > 0.19) by gelatinization at all starch concentrations.     

Effect of paclobutrazol on water stress-induced ethylene biosynthesis and polyamine accumulation in apple seedling leaves

Ethylene biosynthesis and polyamine content were determined in [(2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol] (paclobutrazol) pre-treated and non-treated water-stressed apple seedling leaves. Paclobutrazol reduced water loss, and decreased endogenous putrescine spermidine content. Gibberellic acid (GA) counteracted the inhibitory effect of paclobutrazol on polyamine content. Paclobutrazol also prevented accumulation of water stress-induced 1-aminocyclopropane-1-carboxylic acid (ACC), 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), ethylene production and polyamines in apple leaves. α-Difluoromethylarginine (DFMA), but not α-difluoromethylornithine (DFMO), inhibited the rise of putrescine and spermidine in stressed leaves. S-Adenosylmethionine (SAM) was maintained at a steady state level even when ethylene and the polyamines were actively synthesized in stressed apple seedling leaves. The conversion of ACC to ethylene did not appear to be affected by paclobutrazol treatment.     

Oviposition behavior of Anastrepha fraterculus in apple and diel pattern of activities in an apple orchard in Brazil

The oviposition behavior and diel pattern of activities of Anastrepha fraterculus (Wied.) (Diptera: Tephritidae) were observed in an apple orchard and fruit characteristics involved in oviposition preferences were investigated in field cage tests. Fruit size influenced fruit acceptability as an oviposition site by females which did not discriminate among the cultivars Gala, Fuji, and Golden Delicious when same-size fruits were presented simultaneously. Oviposition behavior in apples was basically the same as that described for primary hosts. Hourly census of fly activity indicated that adults did not overnight in the orchard and that they entered the orchard around 1100 h when temperature reached about 21 °C . Bird droppings were an important food item for adults. Behavioral differences between males and females might account for a significant biased sex ratio both in the orchard and at the edge of the native surrounding vegetation. Implications for fruit fly management in Brazilian apple orchards are discussed.     

Identification of spin diffusion pathways in proteins by isotope-assisted NMR cross- relaxation network editing

A new isotope-assisted cross-relaxation editing experiment, [1H-13C]DINE-NOESY[1H-15N]HSQC (DINE = Double INEPT Edited), is proposed. It is based on the selectiveinversion of CH/CH3 or CH2 protons in the middle of the mixing time. The experiment sortsout the spin diffusion paths according to the principal mediators, either the CH/CH3 or theCH2 protons. This is useful in the structure refinement process, as it enables proper alignmentof the aliphatic protons in the vicinity of NH protons.     

Freezing behavior of xylem ray parenchyma cells in softwood species with differences in the organization of cell walls

Summary By cryo-scanning electron microscopy we examined the effects of the organization of the cell walls of xylem ray parenchyma cells on freezing behavior, namely, the capacity for supercooling and extracellular freezing, in various softwood species. Distinct differences in organization of the cell wall were associated with differences in freezing behavior. Xylem ray parenchyma cells with thin, unlignified primary walls in the entire region (all cells inSciadopitys verticillata and immature cells inPinus densiflora) or in most of the region (mature cells inP. densiflora and all cells inP. pariflora var.pentaphylla) responded to freezing conditions by extracellular freezing, whereas xylem ray parenchyma cells with thick, lignified primary walls (all cells inCrytomeria japonica) or secondary walls (all cells inLarix leptolepis) in most regions responded to freezing by supercooling. The freezing behavior of xylem ray parenchyma cells inL. leptolepis changed seasonally from supercooling in summer to extracellular freezing in winter, even though no detectable changes in the organization of cell walls were apparent. These results in the examined softwood species indicate that freezing behavior of xylem ray parenchyma cells changes in parallel not only with clear differences in the organization of cell walls but also with subtle sub-electron-microscopic differences, probably, in the structure of the cell wall.     

NMR triple-quantum filtered relaxation analysis of O-water in insulin solutions: an insight into the aggregation of insulin and the properties of its bound water

Transverse triple-quantum filtered NMR spectroscopy (TTQF) of ¹⁷O-water was used to study the properties of water in insulin solutions at different Zn²⁺ concentrations and pH values. It was established that strongly bound water molecules are already present in Zn-free insulin. On the assumption that the effective correlation time of a strongly bound water molecule, τ_sb, is 10 ns, the apparent number of strongly bound water molecules was 3 to 4 per insulin monomer. Addition of Zn²⁺ equivalent to 2 g-atoms per hexamer did not produce substantial increases in the overall ¹⁷O-water TTQF signal intensity and apparent fraction of bound water. The dramatic enhancement of the TTQF signals observed for samples with a Zn²⁺/hexamer ratio greater than 2:1 could be attributed to the increase in correlation time of the strongly bound water, due to the formation of higher-order oligomers of the protein.     

Simplified analysis of acetaminophen glucuronide for quantifying gluconeogenesis and glycogenolysis using deuterated water

Measurement of acetaminophen glucuronide (AG) ²H enrichment from deuterated water (²H₂O) by ²H nuclear magnetic resonance (NMR) analysis of its monoacetone glucose (MAG) derivative provides estimation of gluconeogenic and glycogenolytic contributions to endogenous glucose production (EGP). However, AG derivatization to MAG is laborious and unsuitable for high-throughput studies. An alternative derivative, 5-O-acetyl monoacetone glucuronolactone (MAGLA), was tested. Eleven healthy subjects ingested ²H₂O to 0.5% body water enrichment and 500 mg of acetaminophen. Plasma glucose and urinary glucuronide positional ²H enrichments were measured by ²H NMR spectroscopy of MAG and MAGLA, respectively. A Bland–Altman analysis indicated agreement at the 95% confidence level between glucose and glucuronide estimates.     

Radial diffusion transport of water in various zones of maize root and its sensitivity to mercury chloride

NMR-spin echo method was used for comparative study of radial water diffusion in various zones of maize (Zea mays L., cv. Donskaya 1) primary root. Coefficients of water diffusion varied strongly along the root length; the pattern of variations depended on the period during which the diffusion of water molecules was traced. Water diffusion transport in various root zones was unevently sensitive to mercury chloride, an aquaporin inhibitor. The discovered variations in the mobility of water molecules were assigned to morphological and functional features of cells and tissues in the root zones examined; they were interpreted in terms of variable contribution and redistribution of water flows along several transport pathways. The decrease in diffusional water flows could be caused by cell wall modifications (deposition of suberin) that emerge in the endoderm regions distant from the root apex and diminish the contribution of apoplastic transport.     

Frequency dependence of water proton longitudinal nuclear magnetic relaxation times in mouse tissues at 20°C

We have measured the proton longitudinal relaxation times of tissue water of healthy and tumor-bearing mice as a function of the Larmor frequency in the range 6.7 to 90 MHz. These data can be rationalized according to , where A and B are constants specific to the tissue species. We present an interpretation of this frequency dependence within the Fast Exchange Two States model. It is shown that involving a distribution of correlation times for water proton-proton interaction does not yield consistent results, whereas a physically meaningful translational diffusion model pertinent to the dipolar interaction between water protons and macromolecules protons leads to the required frequency dependence. Essentially tissues would differ by the ‘bound’ versus ‘free’ proportion, or by structural properties of cells, rather than by the time-scales governing water motion.     

Biophysical mechanisms of physiological water exchange with the surroundings by the cells of the <Emphasis Type="Italic">Nitella translucens</Emphasis> and <Emphasis Type="Italic">Chara corallina</Emphasis> plants

In the present study, we discussed the biophysical mechanisms of stationary water exchange with the surroundings by the Nitella translucens and Chara corallina plants. It was postulated that these plants, which subsist under total immersion in a water medium, conduct water exchange within single cells individually. With the application of the equations of mechanistic formalism for membrane transport to our investigations (Kargol and Kargol 2000, Kargol 2001, Kargol 2002, Kargol and Kargol 2003, 2003*), it was demonstrated that individual cells of these plants can simultaneously absorb and remove considerable amounts of water at constant cell volume, i.e. under stationary conditions. Water absorption is osmosis-driven, and its removal is effected by the cell turgor pressure.     

Modelling advection and diffusion of water isotopologues in leaves

We described advection and diffusion of water isotopologues in leaves in the non-steady state, applied specifically to amphistomatous leaves. This explains the isotopic enrichment of leaf water from the xylem to the mesophyll, and we showed how it relates to earlier models of leaf water enrichment in non-steady state. The effective length or tortuosity factor of isotopologue movement in leaves is unknown and, therefore, is a fitted parameter in the model. We compared the advection-diffusion model to previously published data sets for Lupinus angustifolius and Eucalyptus globulus. Night-time stomatal conductance was not measured in either data set and is therefore another fitted parameter. The model compared very well with the observations of bulk mesophyll water during the whole diel cycle. It compared well with the enrichment at the evaporative sites during the day but showed some deviations at night for E. globulus. It became clear from our analysis that night-time stomatal conductance should be measured in the future and that the temperature dependence of the tracer diffusivities should be accounted for. However, varying mesophyll water volume did not seem critical for obtaining a good prediction of leaf water enrichment, at least in our data sets. In addition, observations of single diurnal cycles do not seem to constrain the effective length that relates to the tortuosity of the water path in the mesophyll. Finally, we showed when simpler models of leaf water enrichment were suitable for applications of leaf water isotopes once weighted with the appropriate gas exchange flux. We showed that taking an unsuitable leaf water enrichment model could lead to large biases when cumulated over only 1 day.     

Antibody-specific detection of caveolin-1 in subapical compartments of MDCK cells

Caveolin-1 is the major structural component of caveolae and is also found in the Golgi complex of many cell types. Occasionally, caveolin-1 has been observed in additional intracellular compartments, including recycling endosomes. Why caveolin-1 expression is detected at these sites only infrequently is not clear. In this study, we test the hypothesis that non-caveolar, non-Golgi pools of caveolin-1 display unique and/or fixation-dependent epitopes. We compared the ability of a panel of antibodies raised against various domains of caveolin-1 to detect distinct subcellular pools of the protein by immunofluorescence microscopy in Madin-Darby canine kidney (MDCK) cells, a cell line where the subcellular localization of caveolin-1 has been extensively characterized. We show that three antibodies directed to the N-terminus of caveolin-1 recognize a previously undetected pool of caveolin-1 in the subapical region of MDCK cells, a localization characteristic of endosomal recycling compartments. The antibodies vary in their ability to label caveolin-1 at the cell surface, and the epitopes detected by each are highly fixation dependent. Our findings suggest that no single caveolin antibody or staining condition is capable of detecting all the caveolin-1 in a cell simultaneously. Consequently, the subcellular distribution of caveolin-1 may be much broader than currently believed.     

Solute concentrations of the phloem and parenchyma cells present in squash callus

Abstract. Glutaraldehyde fixation was used to determine the solute concentrations in the various cell types present in tissue cultures of squash ( Cucurbita pepo ). Small pieces of callus were plasmolyzed in a graded series of mannitol solutions and fixed in 20 kg m⁻³ glutaraldehyde adjusted to be isosmotic with the particular plasmolysing solution. The callus samples were further processed using standard electron microscopy techniques. Using this procedure, mature sieve elements that form in squash callus have an osmotic potentional of -2.4MPa. The osmotic potential of the callus sieve elements was comparable to values reported for the sieve tube members of the phloem in intact plants. This ability of callus sieve elements to develop high internal hydrostatic pressures demonstrates that they are capable of phloem loading. However, the osmotic potentials of the surrounding parenchymatous cells and companion cells were only –1.15 and –1.5 MPa, respectively. In contrast to the companion cells of the phloem in intact plant tissues, the osmotic potential of the callus companion cells indicated that they were not directly involved in phloem loading. Several immature sieve elements containing distinct nuclei and vacuoles were observed in the callus granules. These immature sieve elements were plasmolyzed in weaker mannitol solutions (below 0.6kmol m⁻³) than the enucleate sieve elements (1.01 kmol m⁻³ mannitol). The low solute concentrations in immature sieve elements indicated that the ability to load sugars occurs concomitantly with the maturation of the sieve element protoplast.     

Identification of bound water molecules in the cyclic tetrasaccharide, cyclo-{-->6}-alpha-d-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->)

A structural characterization of bound water molecules in the cyclic tetrasaccharide, cyclo-{-->6}-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->), was carried out by NMR spectroscopy. H-1', 2'-OH, H-3', and 4'-OH of the 3-O-glycosylated residue and H-1 of the 6-O-glycosylated residue were found to cross-relax with protons of bound waters using the double-pulsed field-gradient spin-echo ROESY experiment. In the crystal structure, one water molecule is located in the center of the plate, and its temperature factor is very low, indicating that this water molecule is an intrinsic component.     

Phenotype modulation in primary cultures of rat aortic smooth muscle cells

The transition of adult rat aortic smooth muscle cells from a contractile to a synthetic phenotype during the first week of primary culture on a substrate of fibronectin in serum-free medium was studied by light and electron microscopy. The weak base chloroquine and the carboxylic ionophore monensin were both found to inhibit the spreading of the cells and the accompanying changes in cellular fine structure. The exchange of myofilament bundles for a prominent rough endoplasmic reticulum and Golgi complex was delayed and vacuoles filled with incompetely degraded material accumulated in the cytoplasm. The microtubule-disruptive drugs colchicine and nocodazole likewise opposed the spreading and fine structural reorganization of the cells. Most typically, the Golgi stacks were small and widely dispersed. In addition, vacuoles of the type mentioned above increased in number. On the other hand, there was surprisingly little effect of cytochalasin B, a drug that is supposed to interfere with the assembly of actin filaments. The observations suggest that the phenotypic modulation of arterial smooth muscle cells is dependent on: (a) lysosomal degradation of discarded cellular constituents, (b) active vesicular transport along the exocytic pathway to provide the expanding cell surface with new membrane, and (c) a normal microtubular cytoskeleton to ensure the establishment of a new and functionally efficient intracellular organization.     

Ultrastructural evidence for the presence of secretory cells in the pineal parenchyma ofHeteropneustes fossilis

There is an extensive literature dealing with the study of indoles, especially serotonin and melatonin, but with considerably less emphasis on the cells and cell types involved in the synthetic process. In the present electron microscopical investigation of the pineal end vesicle ofHeteropneustes fossilis, pinealocytes have been revealed in the pineal parenchyma characterized with extensive synthetic apparatus viz., rough endoplasmic reticulum, free ribosomes, lipid droplets, mitochondria and Golgi bodies. Two sub-populations of the pinealocytes are easily distinguishable on the basis of electron opacity and the preponderance of one or other morphological profile: light cells and dark cells. Light cells represent the active phase of secretion while dark cells represent the storage and release phase of secretion. A neuroendocrine role for the pineal body inHeteropneustes fossilis is suggested which may be significant in view of the nocturnal habit of the fish. Dedicated to my father, Prof. C B L Srivastava