Cellular localization of Arabidopsis xyloglucan endotransglycosylase-related proteins during development and after wind stimulation.

A gene family encoding xyloglucan endotransglycosylase (XET)-related proteins exists in Arabidopsis. TCH4, a member of this family, is strongly up-regulated by environmental stimuli and encodes an XET capable of modifying cell wall xyloglucans. To investigate XET localization we generated antibodies against the TCH4 carboxyl terminus. The antibodies recognized TCH4 and possibly other XET-related proteins. These data indicate that XETs accumulate in expanding cell, at the sites of intercellular airspace formation, and at the bases of leaves, cotyledons, and hypocotyls. XETs also accumulated in vascular tissue, where cell wall modifications lead to the formation of tracheary elements and sieve tubes. Thus, XETs may function in modifying cell walls to allow growth, airspace formation, the development of vasculature, and reinforcement of regions under mechanical strain. Following wind stimulation, overall XET levels appeared to decrease in the leaves of wind-stimulated plants. However, consistent with an increase in TCH4 mRNA levels following wind, there were regions that showed increased immunoreaction, including sites around cells of the pith parenchyma, between the vascular elements, and within the epidermis. These results indicate that TCH4 may contribute to the adaptive changes in morphogenesis that occur in Arabidopsis following exposure to mechanical stimuli.     

An unusual electrooptical effect observed for DNA fragments and its apparent relation to a permanent electric moment associated with bent DNA

Dichroism decay curves of DNA fragments with chain lengths in the range of 179-256 bp show an amplitude inversion suggesting the existence of a positive dichroism component, when these fragments are dissolved at monovalent salt concentrations above approx. 5 mM and are exposed to field pulses with amplitudes and/or lengths above critical values. At the critical values, the unusual dichroism is reflected by an apparent acceleration of the decay curves, which can be fitted by single exponentials with time constants much below the values expected from the DNA contour lengths. The critical pulse amplitudes and lengths decrease with increasing DNA chain length and increasing salt concentration. The experimental data are consistent with results obtained by hydrodynamic and electric model calculations on smoothly bent DNA double helices. The DNA is represented by a string of overlapping beads, which is used to calculate the rotational diffusion tensor and the center of diffusion. The distribution of phosphate charges is asymmetric with respect to this center and thus gives rise to a substantial permanent dipole moment. The magnitude of this dipole moment is calculated as a function of DNA curvature and is used together with experimental values of polarizabilities for simulations of dichroism decay curves. The curves simulated for bent DNA show the same phenomenon as observed experimentally. The ionic strength dependence of the unusual dichroism is explained by an independently observed strong decrease of the polarizability with increasing salt concentration. The field strength dependence is probably due to field-induced bending of double helices driven by the change of the dipole moment. Although our calculations are on rigid models of DNA and thus any flexibility of the double helix has not been considered, we conclude that the essential part of our experimental results can be explained by our model.     

UV–Vis spectroscopy of tyrosine side-groups in studies of protein structure. Part 1: basic principles and properties of tyrosine chromophore

Spectroscopic properties of tyrosine residues may be employed in structural studies of proteins. Here we discuss several different types of UV–Vis spectroscopy, like normal, difference and second-derivative UV absorption spectroscopy, fluorescence spectroscopy, linear and circular dichroism spectroscopy, and Raman spectroscopy, and corresponding optical properties of the tyrosine chromophore, phenol, which are used to study protein structure.     

Cytosolic isocitrate dehydrogenase in humans, mice, and voles and phylogenetic analysis of the enzyme family

In this study, we report cDNA sequences of the cytosolic NADP-dependent isocitrate dehydrogenase for humans, mice, and two species of voles (Microtus mexicanus and Microtus ochrogaster). Inferred amino acid sequences from these taxa display a high level of amino acid sequence conservation, comparable to that of myosin beta heavy chain, and share known structural features. A Caenorhabditis elegans enzyme that was previously identified as a protein similar to isocitrate dehydrogenase is most likely the NADP-dependent cytosolic isocitrate dehydrogenase enzyme equivalent, based on amino acid similarity to mammalian enzymes and phylogenetic analysis. We also suggest that NADP-dependent isocitrate dehydrogenases characterized from alfalfa, soybean, and eucalyptus are most likely cytosolic enzymes. The phylogenetic tree of various isocitrate dehydrogenases from eukaryotic sources revealed that independent gene duplications may have given rise to the cytosolic and mitochondrial forms of NADP-dependent isocitrate dehydrogenase in animals and fungi. There appears to be no statistical support for a hypothesis that the mitochondrial and cytosolic forms of the enzyme are orthologous in these groups. A possible scenario of the evolution of NADP-dependent isocitrate dehydrogenases is proposed.      

Electrostatics of hemoglobins from measurements of the electric dichroism and computer simulations.

Hemoglobins from normal human cells, from sickle cells, and from horse were investigated by electrooptical methods in their oxy and deoxy forms. The reduced linear dichroism measured as a function of the electric field strength demonstrates the existence of permanent dipole moments in the range of 250-400 Debye units. The reduced limiting dichroism is relatively small (< or = 0.1); it is negative for hemoglobin from sickle cells and positive for the hemoglobins from normal human cells and from horse. The dichroism decay time constants are in the range from about 55 to 90 ns. Calculations of the electrooptical data from available crystal structures are given according to models of various complexity, including Monte Carlo simulations of proton fluctuations with energies evaluated by a finite difference Poisson-Boltzmann procedure. The experimental dipole moments are shown to be consistent with the results of the calculations. In the case of human deoxyhemoglobin, the root mean square dipole is higher than the mean dipole by a factor of about 4.5, indicating a particularly large relative contribution due to proton fluctuations. The ratio of the root mean square dipole to the mean dipole is much smaller (approximately 1.1 to approximately 1.5) for the other hemoglobin molecules. The calculations demonstrate that the dichroism decay time constants are not simply determined by the size/shape of the proteins, but are strongly influenced by the orientation of the dipole vector with respect to the axis of maximal absorbance. The comparison of experimental and calculated electrooptical data provides a useful test for the accuracy of electrostatic calculations and/or for the equivalence of structures in crystals and in solutions.     

Cellular localization of the Ca2+ binding TCH3 protein of Arabidopsis

TCH3 is an Arabidopsis t ou ch ( TCH ) gene isolated as a result of its strong and rapid upregulation in response to mechanical stimuli, such as touch and wind. TCH3 encodes an unusual calcium ion-binding protein that is closely related to calmodulin but has the potential to bind six calcium ions. Here it is shown that TCH3 shows a restricted pattern of accumulation during Arabidopsis vegetative development. These data provide insight into the endogenous signals that may regulate TCH3 expression and the sites of TCH3 action. TCH3 is abundant in the shoot apical meristem, vascular tissue, the root columella and pericycle cells that give rise to lateral roots. In addition, TCH3 accumulation in cells of developing shoots and roots closely correlates with the process of cellular expansion. Following wind stimulation, TCH3 becomes more abundant in specific regions including the branchpoints of leaf primordia and stipules, pith parenchyma, and the vascular tissue. The consequences of TCH3 upregulation by wind are therefore spatially restricted and TCH3 may function at these sites to modify cell or tissue characteristics following mechanical stimulation. Because TCH3 accumulates specifically in cells and tissues that are thought to be under the influence of auxin, auxin levels may regulate TCH3 expression during development. TCH3 is upregulated in response to low levels of exogenous indole-3-acetic acid (IAA), but not by inactive auxin-related compounds. These results suggest that TCH3 protein may play roles in mediating physiological responses to auxin and mechanical environmental stimuli.     

Methyl esterification of m7G5′p reversibly blocks its activity as an analog of eukaryotic mRNA 5′-caps

The methyl ester of m⁷G^5′ p was synthesized by a carbodiimide-catalyzed reaction of G^5′ p with methanol followed by dimethylsulfate alkylation. Comparative spectral analyses indicated that m⁷Gp · methyl ester retained the rigid conformation characteristic of the messenger RNA cap analog, m⁷G^5′ p but not its strong inhibitory activity against initiation of capped mRNA translation. Attachment of reovirus mRNA to wheat germ ribosomes, crosslinking of capbinding protein to the 5′-end of oxidized mRNA, and stimulation by this protein of capped mRNA translation in HeLa cell extract were all several-fold more sensitive to inhibition by m⁷G^5′ p than to m⁷Gp · methyl ester. Conversion of the esterified analog to m⁷G^5′ p by digestion with venom phosphodiesterase restored completely the ability to inhibit initiation complex formation. The results indicate that structural features of the 5′-terminal m⁷G cap of mRNA over and above preferred conformation are recognized during eukaryotic protein synthesis.     

Human skeletal muscle: participation of different metabolic activities in oxidation of L-lactate.

The pure mitochondrial fraction obtained from human skeletal muscle did not show coupled L-lactate (+ NAD) oxidation, but this function could be restored by addition of LDH. Thus the "direct", coupled oxidation of L-lactate described earlier (Popinigis et al., 1990. International Perspectives in Exercise Physiology, Human Kinetics Books, pp. 132-133) should be attributed to contaminations.     

Effect of HMG-CoA reductase inhibitors on the erythrocyte membrane]

We studied 10 patients affected by primary hypercholesterolemia treated with placebo for 1 month and with simvastatin (20 mg die) for 6 months during a double-blind clinical trial. At 1-month intervals we determined the following parameters in the serum: total and HDL-cholesterol, triglycerides, apolipoprotein A1 and B. At the same time intervals, we also determined the cholesterol and phospholipid concentration, the Na+/K+ ATPase activity and the fluidity of the erythrocyte membranes. Our results demonstrated the following modifications in the erythrocyte membranes during simvastatin treatment: 1) an initial increase in the cholesterol concentration and in the cholesterol/phospholipid ratio, with a significant decrease only after 4 months; 2) a similar behaviour of membrane fluidity, with an initial decrease and an elevation after 4 months; 3) an increase in the Na+/K+ ATPase activity only after 4 months. We hypothesize that simvastatin not only inhibits the hepatic synthesis of cholesterol, but also modifies the cholesterol exchange between plasma and the erythrocyte membrane.     

Analysis of the cell cycle in the root meristem of Allium cepa under the influence of ledakrin

The influence of a polish anticancer drug on the cell cycle using Allium test was studied. Methods of aceto-orcein squash slides, curve of labelled mitoses after 3H-thymidine incubation and cytophotometrics after Feulgen's reaction were employed. Ledakrin acts strongly antimitotically, but it does not block the cell cycle completely. The cytostatic activity of ledakrin results from its action on the interphase. The phases G1 and S are prolonged while M is unchanged after 6h incubation with ledakrin. During postincubation in water without ledakrin it was noted, at the beginning, that the mitotic activity decreases and it is brought about the lengthening of S and G2 phases. The duration of the cell cycle phases returns to the control level during further postincubation. The results of analysis of chromatin aberrations and the micronucleus test point to a mutagenic effect of ledakrin.