Carbon-13 and deuterium isotope effects on the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase

Carbon-13 and deuterium isotope effects have been measured on the reaction catalyzed by rabbit muscle glyceraldehyde-3-phosphate dehydrogenase in an effort to locate the rate-limiting steps. With D-glyceraldehyde 3-phosphate as substrate, hydride transfer is a major, but not the only, slow step prior to release of the first product, and the intrinsic primary deuterium and 13C isotope effects on this step are 5-5.5 and 1.034-1.040, and the sum of the commitments to catalysis is approximately 3. The 13C isotope effects on thiohemiacetal formation and thioester phosphorolysis are 1.005 or less. The intrinsic alpha-secondary deuterium isotope effect at C-4 of the nicotinamide ring of NAD is approximately 1.4; this large normal value (the equilibrium isotope effect is 0.89) shows tight coupling of hydrogen motions in the transition state accompanied by tunneling. With D-glyceraldehyde as substrate, the isotope effects are similar, but the sum of commitments is approximately 1.5, so that hydride transfer is more, but still not solely, rate limiting for this slow substrate. The observed 13C and deuterium equilibrium isotope effects on the overall reaction from the hydrated aldehyde are 0.995 and 1.145, while the 13C equilibrium isotope effect for conversion of a thiohemiacetal to a thioester is 0.994, and that for conversion of a thioester to an acyl phosphate is 0.997. Somewhat uncertain values for the 13C equilibrium isotope effects on aldehyde dehydration and formation of a thiohemiacetal are 1.003 and 1.004.     

Indolacetic and humic acids induce lateral root development through a concerted plasmalemma and tonoplast H<Superscript>+</Superscript> pumps activation

Increasing evidences have indicated that humic substances can induce plant growth and productivity by functioning as an environmental source of auxinic activity. Here we comparatively evaluate the effects of indole-3-acetic acid (IAA) and humic acids (HA) isolated from two different soils (Inseptsol and Ultisol) and two different organic residues (vermicompost and sewage sludge) on root development and on activities of plasmalemma and tonoplast H⁺ pumps from maize roots. The data show that HA isolated from these different sources as well as low IAA concentrations (10⁻¹⁰ and 10⁻¹⁵ M) improve root growth through a markedly proliferation of lateral roots along with a differential activation not only of the plasmalemma but also of vacuolar H⁺-ATPases and H⁺-pyrophosphatase. Further, the vacuolar H⁺-ATPase had a peak of stimulation in a range from 10⁻⁸ to 10⁻¹⁰ M IAA, whereas the H⁺-pyrophosphatase was sensitive to a much broader range of IAA concentrations from 10⁻³ to 10⁻¹⁵ M. It is proposed a complementary view of the acid growth mechanism in which a concerted activation of the plasmalemma and tonoplast H⁺ pumps plays a key role in the root cell expansion process driven by environment-derived molecules endowed with auxinic activity, such as that of humic substances.     

Changes in root development of Arabidopsis promoted by organic matter from oxisols

The presence of ramified and abundant lateral roots is fundamental to plant growth on highly weathered soils. In this work, the effects of humic acid (HA), fulvic acid and hexanic–methanolic (HM) extract, after alkaline extraction from topsoil of seven different oxisols, on the development of roots of Arabidopsis thaliana was evaluated. Furthermore, we used another emergent plant model, that is, micro‐tom (MT) tomatoes with size similar to that of Arabidopsis to test the effects of HA. It was observed that both humic fractions and the HM extract were able to change the root development, improving the number of lateral roots and their development in comparison to control plants. The promotion of root growth by the three organic matter fractions was higher than that observed by 10^?6 mol L^?1 indole acetic acid. The treatment of MT tomato mutant, less sensitive to auxin, with HA did not promote the emergence of lateral roots, being an additional indication of auxin‐like activities of HA. However, some organic matter fractions exhibited, besides promotion of lateral roots number, increase in the length of principal root, which is not a typical auxin effect, indicating that these substances could contain other physiologically active substances.     

Metabolic profile and antioxidant responses during drought stress recovery in sugarcane treated with humic acids and endophytic diazotrophic bacteria

Water deficit is the major yield‐limiting factor for sugarcane crop production that can be enhanced by inoculating with plant growth promoting bacteria (PGPB) combined with humic substances. The aim of this work was to examine changes to the metabolic profile and antioxidant enzyme activity of sugarcane treated with PGPB and humic acid (HA) after drought and then rehydration. The drought was imposed by withholding irrigation for 21 days thereby measuring enzyme activity, metabolic profile and photosynthetic rate 1 week after rehydratation. Growth of plants treated with HA, PGPB and with both treatments combined (PGPB + HA) was higher than control plants. The antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) activities remained higher after rehydration only in plants treated with HA. Plants treated with HA and PGPB + HA exhibited increased transpiration, stomatal conductance and net photosynthesis than plants treated with PGPB. The PGPB‐treated plants exhibited drought resistance that resembled ‘delayed stress onset’, which is a new term for preserving water in the plants tissues. Water preservation in plants treated with PGPB was corroborated by higher relative water content (RWC) than control plants at the end of the drought period. Plants treated with HA + PGPB exhibited the highest water potential after rehydration and high RWC. Osmotic adjustment in the other treatments (control, HA and PGPB) was indicated by a new pattern of metabolic response after rehydration, including generally enhanced carbohydrates and proteins and specific changes induced by HA‐enhancing aromatic compounds, whereas PGPB exhibited enhanced fatty acids and other aliphatic H species. Humic acids assist with drought stress recovery by inducing antioxidant enzyme activity whereas PGPB induced preservation of leaf water potential and RWC by closing stomata efficiently, resulting in plant water preservation.     

Secretome of HepG2 cells infected with dengue virus: implications for pathogenesis

Viral hemorrhagic fever is a clinical syndrome that poses serious global health threat. Among the causative agents, dengue virus (DV) has the highest incidence rate and its infection is the major cause of viral hemorrhagic fever in the world. Although the pathophysiological mechanisms of DV-induced diseases are not yet understood, it is well accepted that liver is a site of viral replication. In this study, we used proteomics to analyze infection of a hepatic cell lineage, HepG2, with DV, focusing on the secreted proteins. 1D-electrophoresis and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) were used, allowing the identification of a total of 107 proteins, among which 35 were found only in control secretome and 24 only in infected cells secretome. To validate these data, we performed 2D-eletrophoresis followed by MALDI-TOF/TOF, resulting in the identification of 20 proteins, 8 of them confirming LC-MS/MS results. We discuss the results obtained taking into account the proteins previously described in the secretome of HepG2 cells, proteins present in human plasma and proteins of interest for dengue pathogenesis. Altogether the data presented here provide clues for the progress in the understanding of the role of liver secretion in the progression of the disease.     

Slow Transients in the Activity of the NAD Malic Enzyme from Crassula

The NAD malic enzyme from Crassula argentea shows a slow reaction transient in the form of a lag before reaching a steady-state rate in assays. This lag, which has a half-time or τ ranging from seconds to many minutes under various conditions, poses problems in the interpretation of kinetic data with this enzyme. The NAD malic enzyme from Kalanchoë daigremontiana has a similar lag.     

Kinetic properties of NAD malic enzyme from cauliflower

The kinetic characteristics of NAD malic enzyme purified to homogeneity from cauliflower florets have been examined. Free NAD⁺ is the active form of this coenzyme. Double-reciprocal plots of data obtained by varying NAD⁺ and malate^2? at a saturating concentration of Mg²⁺ or by varying Mg²⁺ and NAD⁺ at a saturating level of malate^2? are of intersecting type. This indicates that NAD malic enzyme obeys a sequential mechanism. Analysis of these sets of data suggests that each of these substrate pairs binds randomly to the enzyme. However, each substrate binds tighter when others are already present on the enzyme. NAD malic enzyme cannot decarboxylate malate^2? in the absence of either Mg²⁺ or NAD⁺. Arrhenius plots of the NAD-linked reaction are concave downward, indicating the existence of two rate-determining steps with activation energies of 26.5 and 14.2 kcal/mol, respectively. In addition to Mg²⁺, the enzyme can also use Mn²⁺ and Co²⁺. Using Co²⁺ in place of Mg²⁺ does not change V_max or K_m,malate^2? but the K_m for metal and NAD⁺ are greatly decreased. At pH 7.0 and above, Mn²⁺ isotherms and malate^2? curves with Mn²⁺ are nonlinear and appear to be composed of two separate saturation curves. NAD malic enzyme is completely and irreversibly inactivated by N-ethylmaleimide. The enzyme is also irreversibly inactivated approximately 50% by KCNO.     

Humic acids isolated from earthworm compost enhance root elongation,lateral root emergence,and plasma membrane H+-ATPase activity in maize roots

Earthworms (Eisenia foetida) produce humic substances that can influence plant growth by mechanisms that are not yet clear. In this work, we investigated the effects of humic acids (HAs) isolated from cattle manure earthworm compost on the earliest stages of lateral root development and on the plasma membrane H(+)-ATPase activity. These HAs enhance the root growth of maize (Zea mays) seedlings in conjunction with a marked proliferation of sites of lateral root emergence. They also stimulate the plasma membrane H(+)-ATPase activity, apparently associated with an ability to promote expression of this enzyme. In addition, structural analysis reveals the presence of exchangeable auxin groups in the macrostructure of the earthworm compost HA. These results may shed light on the hormonal activity that has been postulated for these humic substances.